Analysis of Xbp-mRNA RT-PCR. Polymerase Chain Reaction (PCR)
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Transcript of Analysis of Xbp-mRNA RT-PCR. Polymerase Chain Reaction (PCR)
Reverse Transcription of Target RNA
• Uses “downstream or “right primer”
• Extends from target right end (3’) to left end (5’)
• Uses DNA nucleotides
• Creates a “First Strand” of cDNA
• First strand is copied from left primer to give double stranded DNA
Designing Primers for RT-PCR for Xbp-1 mRNA Analysis
• Criteria– Must bracket target sequence in mRNA– Must be at least 17 NT long– Must have a G+C content of ≈ 50-60%– Should have 5’ “GC clamp”– Should not dimerize– Should not form hairpin structures
• WormBase Sumary for xbp-1 gene
– Brief ID: xbp-1 encodes a bZIP transcription factor orthologous to yeast Hac1 and mammalian X box-binding protein 1 (XBP-1, OMIM:194355); XBP-1 is required for the unfolded protein response (UPR) that counteracts cellular stress induced by accumulation of unfolded proteins in the endoplasmic reticulum (ER); XBP-1 mRNA is spliced by the IRE-1 endoribonuclease to promote translation of transcriptionally active XBP-1 that positively regulates UPR gene expression to maintain ER homeostasis and promote normal development. [details]
– Species: Caenorhabditis elegansCommon name: xbp-1 (CGC approved)
– Gene model(s): Gene ModelStatusRemarkNucleotides (coding/transcript)ProteinSwissProtAmino AcidsR74.3confirmed by cDNA(s)C. elegans XBP-1 protein; contains similarity to Interpro domains IPR004827 (Basic-leucine zipper (bZIP) transcription factor), IPR008917 ()795/1747 bpWP:CE01056Q22027264 aa
Analysis of PrimersLeft Primer #1 5’ GCAAAGAGAACGAACGACTGAATCAT
GC%= 39.13 TM = 58.2 Forms 1 low stability dimer
Left Primer #2 5’ GAACCAGAGAACGAGTCCG
GC% =60 TM =60.39 No dimers
Right Primer 5’ TGTTCGAGGGTCTCCATCTTCTT 3’ (Reverse compliment of sense strand)
GC% = 45.45 TM = 58.09 Forms one low stability dimer