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Sample preparation for real-time PCRGuidelines for preparing nucleic acids

Contents

Introduction ................................................................................................................................................ 3

Sample requirements for real-time PCR applications ............................................................................ 4

Methods for isolating nucleic acids .......................................................................................................... 5

Isolation of RNA and DNA ......................................................................................................................... 7

Tips for isolating and handling DNA and RNA ........................................................................................12

Storing nucleic acids .................................................................................................................................15

Variables affecting real-time PCR success .............................................................................................16

Nucleic acid quantity and integrity ...........................................................................................................17

Terminology ...............................................................................................................................................19

Ordering information ............................................................................................................................... 20

Sample requirements for real-time PCR

Introduction

Introduction

Real-time PCR Real-time quantitative PCR, also known as qPCR, combines PCR amplification and detection into a single step. This eliminates the need to detect products using gel electrophoresis, but more importantly, enables the method to be truly quantitative. With qPCR, fluorescent dyes are used to label PCR products during thermal cycling. Real-time PCR instruments measure the accumulation of fluorescent signal during the exponential phase of the reaction for fast, precise quantification of PCR products and objective data analysis.

Gene expression analysis Gene expression or messenger RNA (mRNA) analysis is the most commonly used application for qPCR, microarrays, and next-generation sequencing. For the latter two, researchers must isolate RNA and reverse-transcribe it in order to get reliable gene expression results.

miRNA analysis Noncoding RNAs (ncRNAs), such as microRNA (miRNA) and other small ncRNAs, are the focus of one of the most exciting new areas of molecular biology. More and more experimental evidence demonstrates that they play a central role in how the genome is regulated, and how traits are passed on or eliminated by environmental and genetic factors. Scientists have just begun to understand this natural “gene silencing” mechanism and its role in genomic pathways.

Genetic variation analysis Real-time PCR is commonly used for analysis of genomic variations. These techniques are not quantitative and for genotyping using allele-specific TaqMan® Assays, data are collected after thermal cycling is complete (not in real-time as the reaction progresses). Because these applications require a real-time PCR system, rather than a simple thermal cycler, in practice, they are typically considered real-time PCR applications. These analyses include SNP genotyping and rare allele detection. Copy number variation (CNV) analysis is a quantitative method to determine copy number changes in a genome. High-resolution melt (HRM) analysis can also be used to determine genetic variations. HRM analysis is a post-PCR method for discriminating small sequence differences among PCR products. Unlike other qPCR-based methods for genomic DNA analysis, HRM analysis does not require that the exact sequence of the variants are known, so it can be used to identify samples with uncharacterized sequence differences.

Pathogen detection Scientists will typically be amplifying target nucleic acid sequences from selected microbes present in samples collected from complex biological environments.

Sample preparation for real-time PCR | 3

Sample requirements for real-time PCR applications

Table 1. Sample requirements for analysis of mRNA, microRNA, and noncoding RNA.

Type of RNA analysis Sample type Required quantity*

mRNA: TaqMan® Assay– based detection

Total RNA 1 to 100 ng

mRNA: SYBR® Green dye–based detection

Total RNA, depleted of DNA 10 pg to 20 ng

MicroRNA Total RNA, including small RNAs 1 to 10 ng

Noncoding RNA Total RNA, including small RNAs 1 to 100 ng

*Requirements per 20 μL reaction

Table 2. Sample requirements for analysis of DNA.

Application/technique Sample type Required quantity

SNP genotyping(allelic discrimination)

Genomic DNA 1 to 20 ng

High-resolution melt (HRM) Genomic DNA/PCR product 10 to 20 ng

Copy number variation Genomic DNA 10 to 20 ng

General guidelines The type and amount of nucleic acid sample needed for common real-time PCR applications are provided in the tables below.

RNA analysis

DNA analysis

4

Methods for isolating nucleic acids

Methods for isolating nucleic acids

In many situations, any of several nucleic acid isolation options will provide high purity material suitable for analysis using real-time PCR. Alternatively, you may be able to avoid nucleic acid isolation altogether and use sample lysates directly in PCR or reverse transcription (RT)-PCR. The TaqMan® Cells-to-CT

™ kits and the Blood-to-CT

™ Kit are designed for analysis of RNA in sample lysates using real-time RT-PCR and the TaqMan® Sample-to-SNP™ Kit is designed for SNP genotyping using a similar workflow that circumvents DNA purification.

The main factors to consider when deciding which nucleic acid isolation strategy to choose:

• Nucleic acid type needed for the application• Sample type and size• Throughput needs (number of samples to be processed)• Technology preference (e.g., magnetic beads, organic extraction, or silica

filters)

If you are working with soft, unfrozen animal tissues such as liver, or cultured cells, most nucleic acid isolation methods will provide good yields of high-quality nucleic acids. For analyzing microRNA or other small RNA species, however, it is important to choose a method that recovers small molecules. Similarly, sample types that present physical or biochemical challenges for RNA isolation require sample preparation procedures tailored to overcome these challenges; for example hard or fibrous tissues, extremely small samples, or samples containing large amounts of highly expressed RNAs or biomolecules that copurify with nucleic acids.

Formalin-fixed, paraffin-embedded (FFPE) samples are typically archived histology/pathology samples, and have an increasing use and popularity in molecular research. When working with FFPE samples, it is important to remember that they are preserved using heavy processing and harsh fixation, which causes extensive fragmentation of the nucleic acid and can leave “modifications” or “crosslinks” covalently bound to the bases. In addition, there is a large variation between different FFPE samples (including samples with the same tissue type), with a wide range of yield and functionality of the nucleic acids. As a result, extraction and analysis of nucleic acid from FFPE samples is challenging and requires specially developed protocols. Optimal isolation of nucleic acid from FFPE samples first requires removal of the paraffin (using xylene, heat, or another method) followed by enzymatic digestion to release the nucleic acid for filter-based or bead-based purification.

Note that the choice of methodology can have a profound impact on the time and effort required to complete your experiments (Table 3). This can be important when many samples must be processed, or when personnel have limited time and/or training.


Table 3. Overview of nucleic acid isolation technologies.

Isolates Method Product Description Advantages Time*

RNA and DNA Organic solvent

• TRIzol® Reagent

TRIzol® Reagent is a monophasic solution of phenol and guanidine isothiocyanate. Samples are lysed in the reagent, then mixed with chloroform and centrifuged. RNA can be recovered from the aqueous phase, and DNA can be recovered from the interphase and organic phase.

• Scalable, economical method that provides highly pure nucleic acids

• Good for difficult samples, such as fatty or nuclease-rich tissues

2 hr

RNA and DNA Silica filter • PureLink® kits Sample lysis in a chaotropic salt solution, then silica-based filtration to trap and purify nucleic acids

• Fast, easy method suitable for most sample types

• Rapid sample processing and an easy, familiar protocol

45 min

RNA and DNA Combinationmethods

• TRIzol® Plus kits

• RiboPure™ kits

Combination of organic extraction plus column or bead-based purification

• Best nucleic acid purity, eliminates inhibitors

• Good for difficult samples, such as fatty or nuclease-rich tissues

60 min

RNA and DNA Magnetic beads

• MagMAX™ kits• Dynabeads®

kits

Nucleic acids are immobilized on magnetic beads for washing and elution.

• Flexible, scalable throughput• Easily automated for higher

throughput• Low elution volume for more

concentrated RNA

45 min

RNA and DNA Direct lysis • Cells-to-CT™

kits• Blood-to-CT

™ kits

• Sample-to-SNP™ kits

Nucleic acids are not purified; instead samples are lysed in a solution that can be used directly in real-time PCR or RT-PCR.

• Simplest and fastest protocol• Minimal sample handling/loss

7 min

DNA Ion-exchangeligand

• ChargeSwitch® kits

Samples are lysed and added to a ChargeSwitch® 96-well plate or mixed with magnetic ChargeSwitch® beads. Nucleic acids are bound by the ChargeSwitch® ligand and other biomolecules are washed away. Nucleic acid is eluted with a buffer that raises the pH and neutralizes the positive charge.

• Procedure does not include organic reagents or chaotropic salts

• In 96-well plate format, there are no sample transfers

30 min

DNA Organic solvent

• TaqMan® Sample-to-SNP™ Kit

Combines DNA extraction and TaqMan® genotyping: DNA Extract All Reagents kits provide PCR-ready DNA from a wide variety of sample types ranging from blood to buccal swabs to plant tissues. The TaqMan® GTXpress™ Master Mix then provides robust PCR amplification of extracted DNA.

• Fast: Raw biological samples to SNP genotype results, typically in less than 1 hour

• Robust: Universal protocol for all sample types

5 min

*Approximate processing time for ~12 samples.

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Isolation of RNA and DNAIsolation of RNA and DNASuccessful real-time PCR starts with high-quality nucleic acids. In the tables below we provide our suggestions on kits for real-time PCR applications. Isolation kit recommendations are grouped by nucleic acid type:

• Total RNA extraction—Table 4• mRNA isolation—Table 5• miRNA and other small RNA extraction—Table 6• Genomic DNA isolation—Table 7

With some samples it will be necessary to isolate both RNA and DNA. We provide options and tips for those preparations.

For more information on RNA isolation products, please go to lifetechnologies.com/naprep

Table 4 . Recommended kits for total RNA extraction.

Gold standard for highly pure, intact RNA

High-quality RNA typically in less than

20 min

High-throughput purification of RNA and

DNA

Complete, no purification system for qRT-PCR

results

TRIzol® Reagents PureLink® Kits MagMAX™ Kits Cells-to-CT™ Kits

Isolation methodHigh-purity organic

extraction (requires alcohol precipitation)

Fast, convenient silica column

Scalable, flexible format with magnetic beads

Chemical lysis (time to qRT-PCR results)

Prep time ~1 hr < 20 min ~45 min 10 min

Compatible sample types

Most sample types, particularly more difficult

to lyseCells and tissues Cells, blood, plants Cells

Amount of starting material

100 mg of tissue or 107 cells (requires 1 mL reagent)

Up to 200 mg tissue, 5 x 106 to 1 x 108 cells

Up to 100 mg tissue, up to 5 x 106 cells 1–100,000 cells

Reverse transcription reagents included ✔

qPCR master mix included ✔

High throughput compatible

✔ (plate format available) ✔ ✔


Table 5. Recommended kits for mRNA isolation.

Rapid mRNApurified from total RNA

No purification needed, mRNA direct from crude

samples

mRNA from micro-sized

samples. Best for next-generation sequencing of

mRNA

HTP mRNA directly or enrich from

total RNA

Dynabeads® mRNA Purification Kit

Dynabeads® mRNA DIRECT™

Dynabeads® mRNA DIRECT™ Micro Kit mRNA Catcher™ Plus Kit

Prep time 15 min 15 min 15 min 1.5 hr

Direct isolation of mRNA from crude samples

No Yes Yes Yes

mRNA Enrichment from total RNA Yes No No Yes

Amount of starting material < 75 µg total RNA

<20 x 106 cells 2–200 mg animal tissue 4–400 mg plant tissue

<1 x 104 cells <5 mg plant or animal

tissue

100 to 1 x 106 cells <4 mg tissue

40 µL whole blood 100 ng–100 µg Total RNA

FormatFast magnetic bead

capture from pure total RNA

Fast magnetic bead capture directly from crude

samples

Fast magnetic bead capture directly from micro-size samples

HTP-ready 96-well coated plate

High throughput compatible No No No Yes

Kit size 2 mL oligo(dT) beads 5 mL or 10 mL oligo(dT) beads

2 mL oligo(dT) beads(sufficient for 100 mRNA

preps)96 or 960 preps

Table 6. Recommended kits for miRNA and other small RNA extraction.

Rapid purification for enrichment of small

RNAs only30 min isolation from

most samples

Purification for enrichment of

specific small RNAs

High-throughput, pure, concentrated

microRNAs

Complete kit, sample-to-qPCR

from cells

PureLink® miRNA Isolation Kit

mirVana™ miRNA Isolation Kit (with or

without phenol)

TaqMan® ABC miRNA Purification Kit

MagMAX™ for Microarrays Total RNA Isolation Kit

TaqMan® microRNA Cells-to-CT

™ Kit

RNA types isolated Small RNAs only Small and large RNA molecules Small RNAs only Small and large RNA

moleculesSmall and large RNA

molecules

Prep time 20 min 30 min 75 min <1 hr 10 min

Compatible sample types

Fresh or frozen tissue, tissue stored in RNAlater®, cells

Most sample types Fresh or frozen tissue, blood, cells

Fresh or frozen tissue, tissue stored in RNAlater®, cells

Cells

Isolation method Fast, convenient silica column

Highest purity and convenience;

includes both organic extraction and silica

column

10–50 μL blood-related samples;

5 x 104 cultured cells or 1–10 mg tissue

Scalable, flexible format for highest purity; combines

organic extraction with magnetic bead–

based purification

Cells-to-CT™ direct

lysis, no purification required

Reverse transcription reagents included

No No No NoTaqMan®

MicroRNA Reverse Transcription Kit

qPCR master mix included No No No No TaqMan® master mix

Kit size 25 preps 40 preps 40 preps 96 preps100 lysis reactions

+ reagents for 200 qRT-PCR assays

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Isolation of RNA and DNATable 7. Recommended kits for genomic DNA isolation from tissues and cells.

Process the largest amount of tissue

Rapid processing for scale and automation

Most versatile; able to handle variety of sample types

DNAzol® Reagent MagMAX™ DNA Multi-Sample Kit

PureLink® Genomic DNA Kits

Sample types Cells, tissue Tissue, blood, blood cards, buccal cells, urine Cells, tissue, blood, bacteria

Protocol time 10–30 min <45 min <15 min

Starting material 1–3 x 107 cells25–50 mg tissue

<50 mg of tissue, 50 µL blood, 1 card, 1 buccal swab, 400 µL urine

5 x 106 cellsUp to 25 mg tissue

Isolation technology Organic extraction Scalable, flexible format with magnetic beads Silica spin column

High throughput compatible No Yes No (option available)

Product size 100 mL50 preps 96 preps

480 preps

10 preps 50 preps 250 preps

,

Table 8. Recommended kits for RNA isolation from FFPE samples.

Simple, reliable, rapidEasy to use, highest RNA yields,

tough samplesHTP easy to use, highest RNA

yields, tough samples

PureLink® FFPE RNA Isolation Kit

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE

MagMAX™ FFPE Total Nucleic Acid Isolation Kit

Expected RNA yield per 10 µm section 1 µg Varies,

avg 3.5 µgVaries,

avg 3 µg

Prep time 30 min 2 hr 3 hr (for 96 preps)

Final product extracted Total RNA Total RNA, microRNA, gDNA Total RNA, microRNA, gDNA

Requires organic solvent (xylene) for deparaffinization ✔

High throughput compatible ✔Compatible FFPE sample range Up to ten 10 µm sections Up to four 20 µm sections Up to two 10 µm sections

Kit size 50 rxns 40 rxns 96 rxns

For a complete list of DNA isolation kits, please go to lifetechnologies.com/gDNAprep


Isolation of both RNA and DNA from samples In some cases, it may be desirable to obtain both RNA and DNA from a single

sample. The following products are recommended for this application:

• MagMAX™ Total Nucleic Acid Isolation Kit• MELT™ Total Nucleic Acid Isolation System• RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE

In general, we recommend using purified RNA for real-time PCR applications; however, in some cases good results can be obtained using total nucleic acid samples that contain both RNA and DNA. (Note that purified DNA is required for use with TaqMan® Copy Number Assays.) Here, we discuss potential concerns with using total nucleic acid samples rather than purified RNA or DNA for real-time PCR applications.

Expression analysisTypically, purified RNA is used for analysis of mRNA and small RNA expression. This is because gDNA can participate in reactions in which the qPCR primers do not span an exon–exon junction. Even when primers are designed to span exon junctions, there may be pseudogenes in genomic DNA that can be amplified in mixed nucleic acid samples. Running no-RT controls (reactions that include a mock reverse transcription step) can help to identify gDNA amplification. Another concern with using total nucleic acid samples is the potential that reaction efficiency could be compromised. Certain TaqMan® Assay design includes steps to avoid gDNA detection. (An “_m1” assay is one in which the probe spans an exon–exon junction and should not detect gDNA. An “_s1” assay is one in which the primers and probe lie within a single exon. This assay will detect gDNA. In a “_g1” assay, the assay probe spans an exon junction and will detect gDNA due to off-target mapping. Or, the assay primers and probe lie within a single exon and will detect gDNA due to off-target mapping.) If you design real-time PCR primers/probes yourself, comparing primer/probe sequences to the transcriptome and genome assembly helps ensure that off-target gDNA amplification does not interfere with your analysis.

SNP genotyping and HRM analysisSince RNA is not amplified by most thermostable DNA polymerases, total nucleic acid samples (containing both DNA and RNA) can typically be used for SNP genotyping and high-resolution melt (HRM) DNA analysis by real-time PCR. However, accurate DNA sample quantitation is important for genotyping applications, and RNA will inflate sample quantitation by UV fluorescence. Likewise, DNA will interfere with the accuracy of RNA quantitation. Other methods of DNA quantitation, such as Quant-iT™ DNA assays for the Qubit® Fluorometer, which use DNA-specific dyes, provide accurate DNA quantitation even when there is an equal amount of RNA in the sample. Quant-iT™ assays for RNA and protein are also available.

Copy number analysisThe analysis of copy number variations (CNVs) and smaller genomic regions using TaqMan® Copy Number Assays requires purified DNA for optimal results; total nucleic acid preparations are not recommended for use with these assays.

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Isolation of RNA and DNA

Removal of DNA from RNA samples Generally, RNA isolation protocols favor the recovery of RNA over DNA; many

also include a DNase treatment to remove residual DNA from the preparation. It is not always strictly necessary, but many researchers feel it is prudent to either use a nucleic acid isolation method that includes a DNase treatment step or to treat purified RNA samples with DNase for real-time RT-PCR applications. The product recommendation tables in this technical note indicate which RNA isolation products include a DNase treatment. In addition, we offer several products for removing DNA from prepared RNA samples:

• TURBO DNA-free™ Kit—Hyperactive TURBO™ DNase and reaction buffer for efficient, complete digestion of DNA, plus a novel DNase Inactivation Reagent that removes the DNase and divalent cations from sample without heating, phenol extraction, or precipitation.

• DNA-free™ DNase Treatment & Removal Reagents—Certified RNase-free recombinant DNase I and reaction buffer designed to remove DNA contamination from RNA samples. A novel DNase Inactivation Reagent is also included for rapid reaction cleanup without the risks and hassles of heating, phenol extraction, or alcohol precipitation of samples.

• PureLink® DNase—Designed to be used specifically for on-column digestion of DNA during RNA purification using the PureLink® RNA Mini Kit.


Tips for isolating and handling DNA and RNA

Prevent contamination with PCR products Because real-time PCR is such a sensitive technique, it is subject to

contamination with DNA from completed reactions. Use the following precautions to minimize sample contamination and PCR product carryover:

• Wear a clean lab coat (not previously worn while handling amplified PCR products or used during sample preparation), and clean gloves when preparing samples for PCR amplification. Change gloves whenever you suspect that they are contaminated.

• Maintain separate areas, dedicated equipment, and supplies for: – Sample preparation – PCR setup—never bring amplified PCR products into the PCR setup area – Analysis of PCR products

• Use a set of qPCR reagents that contain UNG in order to digest completed PCR products. These qPCR reagents contain dUTP, which is incorporated into the PCR product and becomes a substrate for the UNG enzyme.

• Open and close all sample tubes carefully, to minimize the chance of contamination. Avoid splashing or spraying PCR samples.

• Use positive-displacement or air-displacement pipettors with filter-plugged tips. Change tips after each use.

• Keep reactions and components capped as much as possible.• Clean lab benches and equipment periodically with 10% bleach solution or

70% ethanol.

Thoroughly disrupt samples Cellular disruption is one of most critical steps affecting the yield and quality

of nucleic acids isolated from biological sources. Disruption needs to be fast and thorough, so that endogenous nucleases are inactivated by exposure to the lysis solution as they are released from their cellular compartments. Complete disruption also optimizes nucleic acid yield. Table 9 shows some general guidelines for appropriate disruption of common sample types.

Table 9. Guidelines for disruption of different sample types.

Soft tissues Hard tissuesCultured cells, blood Bacteria Yeast Fungi

Dounce or polytron ✔ ✔ ✔

Grind in liquid nitrogen with mortar and pestle ✔ ✔ ✔

Pipette vigorously ✔

Bead mill ✔ ✔

Freezer mill ✔

Sonication ✔

Enzymatic disruption ✔ ✔ ✔

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Tips for isolating and handling DNA and RNA

Use protocol modifications when required

Successful analysis of FFPE samples with qRT-PCR and qPCR requires protocol adjustments to manage the fragmentation and modifications of the isolated nucleic acids. These include:

• Targeted amplicon sizes for PCR should be <100 bp to account for the fragmented nature of the nucleic acid.

• Higher inputs of nucleic acids should be used to account for any modifications on the nucleic acid (especially for RNA), which can otherwise lead to a reduction of the reaction efficiency.

• For RNA, heating the samples at a high temperature prior to the reverse transcription step can help relax the modifications and improve efficiency.

Avoid contamination with environmental nucleases

Nucleases, especially RNases, are ubiquitous—they are found in virtually every organism and cell type. In most laboratory settings, however, the primary source of nucleases is laboratory equipment that has been contaminated with DNase or RNase (e.g., from the researcher’s fingertips), or environmental contamination from bacteria, fungi, and their spores. Following are tips for reducing your sample’s exposure to environmental RNases:

• Find a “low-traffic” area that is shielded from air vents and open windows.• Decontaminate benchtop, pipettors, and glassware with RNAseZap®

Solution and Wipes. If you use nucleases in your research (e.g., for plasmid purification), you may want to set up a nuclease-free area for isolating DNA and/or RNA.

• Change gloves frequently (e.g., after touching refrigerator handles, door knobs, equipment, or skin) and wear a clean lab coat.

• Aliquot solutions (including water) into smaller volumes to reduce the risk of contaminating important stock solutions with RNases.

• Use RNase-free tips, tubes, and solutions, and keep them closed whenever possible.

Ambion® RNA essentials for preventing degradation by

RNases Remove RNases from surfaces• RNaseZap® RNase Decontamination Solution (spray or squeeze bottle) and

RNaseZap® Wipes (towelettes) help eliminate RNase contamination from benchtops, glass, and plastic, and can be used to clean work surfaces, pipettors, and equipment. RNaseZap® Solution is a combination of three different chemicals that completely inactivate RNases (and any other enzyme) upon contact.

• ElectroZap™ Electrode Decontamination Solution is a less corrosive formulation used to remove RNase, DNase, and other proteins from electrodes.


Inactivate RNases in solutions• RNAsecure™ Reagent can be used to irreversibly inactivate RNases in

solutions—simply add the reagent and incubate for 10 min at 60°C. This non-enzymatic reagent, which is compatible with RT-PCR and other downstream applications, is ideal for treating primary amine solutions (e.g., Tris). To inactivate new RNase contaminants, simply reheat the solution.

Inhibit RNases in enzymatic reactions• SUPERase⋅In™ Inhibitor—inhibits RNase A type enzymes, RNase T1, and

RNase 1; does not require reducing agents (e.g., dithiothreitol, DTT); works at up to 65°C; effective between pH 5.5 and 8.5

• ANTI-RNase—noncovalently binds neutral pancreatic RNase A type enzymes; does not require reducing agents (e.g., DTT)

• RNase Inhibitor—noncompetitive inhibitor of neutral pancreatic RNase A type enzymes; requires DTT

Nuclease-free buffers, tubes, and tips• DEPC-treated and non–DEPC-treated nuclease-free water, including RT-

PCR–grade water suitable for any PCR application.• Gel running solutions, RNA storage solutions, buffer kit, coprecipitants for

nucleic acid precipitation, and a wide selection of individual buffers.• Certified RNase-free and DNase-free plastics, including barrier and non-

barrier tips, microcentrifuge tubes, PCR tubes, and conical tubes.• RNA ladders and NorthernMax® kits for ultrasensitive northern blot analysis

Inactivate endogenous nucleases immediatelyStart with fresh, high-quality samples and immediately inactivate endogenous nucleases using one of the following methods:

• Thoroughly disrupt samples in lysis solution.• Flash freeze in liquid nitrogen. Tissue pieces must be small enough to freeze

quickly.• Place sample in RNAlater® Tissue Collection: RNA Stabilization Solution.

Tissue pieces must be <0.5 cm in at least one dimension.• Draw blood samples into Tempus® vacutainers to inactivate RNases and to

stabilize RNA profiles. Drawing blood into heparin or EDTA tubes does not achieve this, and can cause a change in profile right after the blood draw.

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Storing nucleic acidsStoring nucleic acids

Proper storage of nucleic acids is critical for successful downstream applications.

Resuspend nucleic acids in a nuclease-free solution with a neutral to slightly acidic pH (pH 6–7) that contains a trace amount of chelating agent (sodium citrate or EDTA) to protect against degradation by introduced nucleases and divalent cations. Resuspension in RNAsecure™ Reagent can increase the stability of RNA.

Divalent ions can catalyze nonspecific RNA cleavage, especially when the solution is heated, that is why we recommend including a chelating agent in nucleic acid storage solutions. One option is to use The RNA Storage Solution, which has greater stability over TE or EDTA and is certified RNase-free.

Short term: Store at –20°C

Long term: Store at –80°C as a precipitate in ethanol, or simply in dilute, nuclease-free buffer.

Aliquot your RNA into several tubes to prevent RNA damage from multiple freeze-thaw events and to help avoid accidental RNase contamination from handling the tube.

While DNA is more stable than RNA we still recommend a neutral pH storage solution and storing at –20°C. Alternatively, DNA can be stored at room temperature if put into the PureLink® DNA stabilization reagent. This is a novel water-free reagent that prevents DNA degradation.


Variables affecting real-time PCR success

Two important variables that affect the performance of qRT-PCR are the presence of inhibitors and the presence of genomic DNA. Inhibitors can affect enzyme performance and can lead to a decrease in sensitivity. gDNA in the qRT-PCR reaction can cause false positive results and background. Another less-understood variable in qRT-PCR performance is RNA quality.

Inhibition of qRT-PCR can decrease the sensitivity of detection, resulting in false negative results, but can also result in the inability to accurately quantitate the RNA of interest.

Most often, inhibition is due to the carryover of chemicals or compounds from the sample preparation process. Examples of some common inhibitors are heme, detergents, and salts. Strict adherence to the sample preparation process, including washes, can reduce the chance that inhibitors are carried over. For some sample preparation reactions, reagents are left in the sample, which do not have an effect at low concentrations but at higher concentrations can cause inhibition. Transfer of too much sample into the qRT-PCR reactions can result in inhibition via this mode.

Monitoring for inhibition is potentially important for every sample that is analyzed and can be done using a variety of tools. One such tool is the Xeno™ RNA control. This is an exogenous RNA that is not similar to any sequence present in available databases. This RNA can be added to the reverse transcription reaction and then detected during qPCR. Identical results for Xeno™ RNA in reactions with and without sample can confirm the lack of inhibitors. An increase in Ct for both Xeno™ RNA and a target confirm that inhibitors are present (Figure 1).

Research samples commonly assayed for viruses include blood, dirt, and tissues. Buffer components and proprietary additives in the many master mixes (e.g., TaqMan® Fast Virus 1-Step Master Mix) have been optimized to handle RT-PCR inhibitors, to help ensure consistent performance even with these difficult samples.

Figure 1. Use of Xeno™ RNA to assess qPCR reactions for inhibitors. (A) A titration of two different cell types prepared using the Cells-to-CT

™ kit protocol. Xeno™ RNA, an exogenous control RNA, is added to the RT reactions. qPCR was performed to assay two genes of different expression levels (ACTB and VEGF) and Xeno™ RNA. The linearity of the target mRNA transcript signal and the maintenance of the Ct for Xeno™ RNA indicate that, as cell number increases, no inhibition occurs. (B) In a similar experiment using a different cell line (known to carry qRT-PCR inhibitors), neither the linearity constant value of the Xeno™ RNA signal nor the linearity of the target mRNA signal were maintained, showing that, as cell number increases, inhibition of the qRT-PCR reactions also increases.

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Nucleic acid quantity and integrityNucleic acid quantity and integrity

Quantitation Note that some real-time PCR applications, such as gene expression analysis, do not require nucleic acid quantitation prior to real-time PCR; internal standards can be used to normalize results. For other applications, such as SNP genotyping, accurate DNA quantitation is important for good results.

There are three common methods used for nucleic acid quantitation:

• Fluorescent dyes—Exhibit a large fluorescent enhancement when bound to nucleic acids. For example the Quant-iT™ assays for the Qubit® Fluorometer include fluorescent dyes specific for DNA, RNA, or protein. The system provides much higher accuracy than UV spectroscopy readings, which cannot distinguish between different macromolecules. Upon binding to DNA, RNA, or protein, the fluorescence of the Quant-iT™ dyes increases several hundred fold, giving a very high signal-to-background ratio. High signal and low background mean exceedingly high sensitivity—up to 1,000 times more sensitive than UV readings.

• UV spectroscopy—Based on absorbance of nucleic acids at 260 nm. For small volumes, the NanoDrop® spectrophotometer is commonly used.

• Agilent® 2100 Bioanalyzer—Uses a combination of microfluidics, capillary electrophoresis, and fluorescent dye that binds to nucleic acid to evaluate nucleic acid integrity and concentration.

Expected nucleic acid yields If you are accustomed to working with tissues where RNA is plentiful, such as liver, you may have unrealistically high expectations of RNA yields from tissues with lower RNA contents (e.g., skin, muscle, bone). Figures 2 and 3 provide general guidelines for estimating RNA yields from a variety of cells and tissues. Remember that RNA yields from analogous tissues in different organisms can also vary.

High yield Moderate yield Low yield

LiverSpleenHeart Kidney

0.05 µg

Bladder

OvaryAdipose

4 µg

Brain

Lung

* µg total RNA/mg tissue. This is intended as a general guide only and may vary depending on physiological state or organism.

Bone

Thymus

Embryo

2 µg

Figure 2. Typical yields of total RNA (µg per mg of sample) from various tissues. This is intended as a general guide only, as yields may vary depending on physiological state or organism.


Integrity and quality evaluation Although PCR and RT-PCR are tolerant of partially degraded nucleic acids,

assessing the integrity of RNA and DNA samples is still important when comparing results from more than one sample. Variations in sample integrity could introduce inconsistencies in downstream results.

RNA Integrity Number (RIN) is a commonly used metric that is calculated from Agilent® bioanalyzer/RNA kit data using a free software tool. The RIN is based on the relative sizes of the rRNA peaks as well as any and all degradation products.

UV spectroscopy is a common method used to assess nucleic acid purity. The absorbance at 280 nm provides a measurement of contaminants with peak absorbances at 280 nm (i.e., proteins), so the A260/A280 ratio provides an estimate of nucleic acid purity.

Pure DNA and RNA preparations have expected A260/A280 ratios of ≥1.8 and ≥2.0, respectively. Nucleic acid samples with a significantly lower ratio should be further purified by phenol-chloroform extraction, LiCl precipitation (RNA only), alcohol precipitation/washing, or with an RNA or DNA cleanup system such as the MEGAclear™ Kit.

Degraded RNA (e.g., as found in FFPE samples) can still be analyzed; however, an increase in Ct values should be expected. To decrease this effect, shorter amplicons can be used. Amplicon lengths can be obtained using the TaqMan® Assay search tool found at lifetechnologies.com/ordertaqman. There’s also a very helpful video at the bottom of this page: “Finding the right TaqMan® Gene Expression Assay”

Figure 3. Guide for estimating RNA yields. The expected amount of total RNA and mRNA from tissues and cells will vary depending on tissue or cell type, physiological state, etc.

Tissue/cells → RNA conversions

5 mg of tissueor 2.5 x 106 cells 1/500 wt

10 µg total RNA

8 µgrRNA~80%

0.3 µg mRNA

~3%

Specific mRNA(moderately abundant)

300 pg or 0.6 fmol

~0.1%

Specific mRNA(rare message)

10 fg–10 pg

<0.001%

1 g of tissueor 5 x 108 cells 1/500 wt

2 mg oftotal RNA

1.6 mgrRNA-80%

60 µg of mRNA

-3%

Specific mRNA(moderately abundant)

60 ng or 120 fmol

-0.1%

Specific mRNA(rare message)

2 fg-2 ng

<0.001%

18

Nucleic acid quantity and integrity

Nucleic acid terminology Some of the common terms used to describe different types of nucleic acids are listed here.

Exome—All exons in the genome.

Genome—An organism’s nucleic acid blueprint; all of the DNA (or RNA for many viruses) native to the organism.

Genomic DNA—gDNA—the DNA that makes up an organism’s genome. In eukaryotes, gDNA is differentiated from extrachromosomal DNA found in organelles such as mitochondria.

Messenger RNA—mRNA—RNA that encodes protein. In eukaryotes it is usually characterized by a 5́ methylguanosine cap and a poly(A) tail.

Methylome—The methylated DNA in a sample.

MicroRNA—miRNA—Small (~22 nt) noncoding RNAs that are produced from longer precursors in a two-step process. miRNAs affect the stability and translation of mRNAs with complete or partial sequence homology.

Noncoding RNA—ncRNA—Any RNA that does not direct translation of protein; includes ribosomal and transfer RNAs in addition to small RNAs.

Small interfering RNA—siRNA are double-stranded 19–25 base pair RNA molecules with 2-nucleotide terminal overhangs. siRNA reduce mRNA levels and hence protein expression by causing mRNA degradation. Upon interaction with an mRNA of complete sequence homology, the RISC protein complex cleaves the target mRNA resulting in its rapid degradation. Many researchers use synthetic siRNAs for biological research to exlore the functions of various intracellular RNA.

Small RNA—Any small (~30 nt and smaller) noncoding RNA molecule. Small RNAs are grouped into several classes, such as miRNA, siRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and piwi-interacting RNA (piRNA).

Total nucleic acid—All nucleic acids in a sample: DNA, RNA, and small RNAs.

Total RNA—Technically, total RNA refers to all of the RNA in a sample, but in a practical sense, it often means “all RNA large enough to be recovered using the isolation method”. Many methods include a solid-support binding step or an alcohol precipitation that preferentially recover RNA molecules larger than 50–150 nt.

Transcriptome—All of the RNA transcribed from an organism’s genome.


Ordering information

Product Quantity Cat. No.RNA isolationPureLink® RNA Mini Kit 50 preps 12183-018A

TRIzol® Plus RNA Purification Kit 50 preps 12183-555

MagMAX™-96 Total RNA Isolation Kit 96 rxns AM1830

Dynabeads® mRNA Purification Kit 2 mL 61006

Dynabeads® mRNA DIRECT™ Kit 5 mL 61011

mirVana™ miRNA Isolation Kit 40 preps AM1560

MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit (Compatible with Tempus® Blood RNA Tubes) 96 preps 4451893

MagMAX™ for Stabilized Blood Tubes RNA Isolation Kit (Compatible with PAXgene® Blood RNA Tubes) 96 preps 4451894

Tempus® Blood RNA Tube 50 tubes 4342792

DNA isolationPureLink® Genomic DNA Kit 50 preps K1820-01

PureLink® Genomic Plant DNA Purification Kit 50 preps K1830-01

GeneCatcher™ gDNA Blood Kit 96 preps CS21101

Dynabeads® DNA Direct Blood Kit 100 preps 631-02

MagMAX™-96 DNA Multi-Sample Kit 96 preps 4413021

RNA and DNA isolationMagMAX™ Total Nucleic Acid Kit 100 rxns AM1840

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE 40 preps AM1975

MagMAX™ FFPE Total Nucleic Acid Isolation Kit 96 rxns 4463365

Direct to qPCRTaqMan® Gene Expression Cells-to-CT

™ Kit 100 lysis/500 PCR AM1728

TaqMan® Fast Cells-to-CT™ Kit 100 lysis/500 PCR 4399003

TaqMan® microRNA Cells-to-CT™ Kit 100 lysis/500 PCR 4391848

Fast SYBR® Green Cells-to-CT™ Kit 100 lysis/500 PCR 4402956

Power SYBR® Green Cells-to-CT™ Kit 100 lysis/500 PCR 4402954

Single Cell-to-CT™ Kit 50 rxns 4458237

Stabilized Blood-to-CT™ Nucleic Acid Preparation Kit for qPCR (Compatible with Tempus® Blood RNA Tubes) 200 rxns 4449080

Stabilized Blood-to-CT™ Nucleic Acid Preparation Kit for qPCR (Compatible with PAXgene® Blood RNA Tubes) 200 rxns 4449082

TaqMan® Sample-to-SNP™ Kit 5 mL and 1 mL 100 sample prep/400 PCR 4403313

Controlling DNases and RNasesTurbo™ DNase 1,000 units AM2238

RNAlater® RNA Stabilization Solution 100 mL AM7020

RNaseZap® RNase Decontamination Solution 250 mL AM9780

Nuclease-Free Water 500 mL AM9930

RNase Free Microcentrifuge Tubes 1,000 tubes AM12300

lifetechnologies.com

For Research Use Only. Not for use in diagnostic procedures. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Trizol and DNAzol are registered trademarks of Molecular Research Center, Inc. NanoDrop is a registered trademark of NanoDrop Technologies LLC. © 2014 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. CO25412 0114

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