Can anybody have the correct primer sequences to amplify APRIL gene (TNF-family) for reverse transcription of m-RNA as different articles use different primer sequences as well as the primers for GAPDH gene (used as a control).
Why does this difference happens in primers used?
Your answers are highly appreciated.
OligodT primers bind to mRNA due to the poly A tail of mRNAs and gives you the chance to compare changes of just mRNAs (all in one sample, eg April and gapdh)
Random primers, mostly hexamers to be most “specifically“ unspecific, transcribe all RNAs, not just mRNAs and gives you the chance to compare changes in all sorts of RNAs in one sample.
specific primers,eg for April, just transcribe your April. No chance to view eg housekeeping genes in the sample. But there are downstream applications where this is certainly useful, e.g. creating a plasmid for overexpression from scratch.
I don’t know which exact publications you are referring to, but these are the possibilities for reverse transcription.
hope this made sense. Best, Teresa
Dear Teresa Otto
Thank you for your answer and sorry for this delay in replying to you.
From different articles i searched, i found specific primers for mRNA expression of APRIL (test) and GAPDH (control) genes as follows:
1. The forward primers (GAPDH-Fw: 5’-GTCCATGCCATCACTGCCAC-3’)
The reverse (GAPDH-Rv: 5’-ATGACCTTGCCCACAGCCTT-3’)
APRIL upstream primer was 5'-CCTACGCATTCCTCAACGAA-3'.
APRIL downstream primer was 5'-TAAACTACCTGATCCCAGCA-3'.
Anticipated product was 214 bp.
2. GAPDH upstream primer was 5'-AATCCACCTCTCAACTACC-3'.
GAPDH downstream primer was 5'-CTCCCCACGCAAGCTTAC-3'.
Anticipated product was 359 bp.
Soluble APRIL (sAPRIL) (aa 105–250) was PCR-amplified from appendix cDNA using primers: OS RbsAPRIL 5′-AGATCTGCACTGCCCACCCAGAAA-3′ and
ASRbsAPRIL 5′-CTGCAGTCACAGTTTCACAAACCC-3′.
From https://www.sciencedirect.com/science/article/abs/pii/S2210776217302557
3.
APRIL upstream primer was 5'-CCTACGCATTCCTCAA CGAA-3'.
APRIL downstream primer was 5'-TAAACTACCT GATCCCAGCA-3'.
Anticipated product was 214 bp.
GAPDH upstream primer was 5'-AATCCACCTCTCAAC TACC-3'.
GAPDH downstream primer was 5'-CTCCCCACGC AAGCTTAC-3'.
Anticipated product was 359 bp.
From Article Characteristics of BAFF and APRIL factor expression in multi...
Can you explain why there are differences in different articles in primer sequences used for mRNA expression in APRIL and GAPDH genes?
Well,
it depends on the lab how they do their primers. You can either make up your own primers or take published sequences. Since you have usually a huge amount of mRNA present (the length of the mRNA), varieties of sequences for primers are given.
i prefer to make my own primers since you can actually decide, at what temperature they are annealing in a PCR, how big your products are (359 is at the upper threshold for real time PCR), how your GC amount is Etc. If you just Order published ones without checking them, you sometimes can run into problems.
I suggest you try to make your own primers (pubmed primer-blast for example), so you understand a little bit more what primers are And how they are developed.
Dont forget; the April in publication 2 is the soluble version so they were looking for this specific version, not for the general version.
I am pretty sure there are videos around wich explain how you develop your own primers.
Best,
Teresa
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