Which one is better in synthetic of first strand cDNA during reverse transcription: Revers Primer or Forward primer or both? I believe the Revers one is useful. Is it quite true?
With many Thanks..
Hello,
if you want to use a Gene Specific Primer (GSP) technique instead of random hexamers or oligo-dT to generate the first strand then you must use a reverse primer. Being single-stranded, your mRNA will only bind to the rev.
Two caveat here:
1) when using GSP the efficiency of your cDNA synthesis reaction is heavily dependent on the characteristics of your primer, so you may have to test more than one in order to optimize.
2) If you use GSP you will be able to use your cDNA only on the GSP target gene and nothing else (e.g. no Q-PCR, no amplification of other genes and so on). To target other genes you will have to start back from the original RNA.
Hi, as you most probably know for cDNA synthesis you can also use random primers or Oligo(dT) Primers. so this means you can use one and should worked. Oligo(dT)20 is a homogenous mixture of 20-mer thymidines and normally I use this one for my experiment. then I think if you use rev or fwd primer it just necessary to be specific for your gene.
good luck
Hello,
if you want to use a Gene Specific Primer (GSP) technique instead of random hexamers or oligo-dT to generate the first strand then you must use a reverse primer. Being single-stranded, your mRNA will only bind to the rev.
Two caveat here:
1) when using GSP the efficiency of your cDNA synthesis reaction is heavily dependent on the characteristics of your primer, so you may have to test more than one in order to optimize.
2) If you use GSP you will be able to use your cDNA only on the GSP target gene and nothing else (e.g. no Q-PCR, no amplification of other genes and so on). To target other genes you will have to start back from the original RNA.
If I understood your question correctly, the answer would be yes. You MUST use a reverse primer for the synthesis of the first strand cDNA. This reverse primer can be either gene specific (the reverse compliment of the actual sequence) if you know your target sequence and want to specifically reverse transcribe it as in the case of 5'-RACE, or you can use a general primer either with or without an adapter for the reverse transcription of the whole mRNA pool.
Check page 3 in the following manual for further details about the actual sequences of the primers:
http://www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17335
http://www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17335
Hi,
For cDNA synthesis if you are going down the gene specific priming (GSP) pathway then a a reverse primer is the one to use. You can also use random hexamers or random hexanucleotides to prepare your cDNA. These are oligonucleotide sequences of 6 bases which are synthesised entirely randomly to give a numerous range of sequences that have the potential to anneal at many random points on a DNA or RNA sequence. They act as a primer to commence first strand cDNA synthesis. Random hexamer cDNA synthesis also allows for flexibility with your RT-PCR because you aren't locked in to one specific gene like you are when you are preparing cDNA using a reverse GSP. You can actually target multiple genes from that one cDNA preparation.
We are conducting Real Time PCR here in our Lab. Industrial Microbiology Research Division, Bangladesh Council of Scientific and Industrial Research, Chittagong, Bangladesh.
Yes you are correct.. ...If you are going to amplify a particular gene of intrest,you can use gene specific REVERSE PRIMER for the conversiton of firsr strand cDNA from total RNA of mRNA.
As the others have said, you do need the reverse primer for what you are suggesting. However, I would suggest performing a separate RT reaction, using a mixture of oligo(dT) and random hexamers, so that you get a first-strand reaction that you can use for multiple reactions.
http://www.sciencedirect.com/science/article/pii/S0003269703005189
Dear John T Corthell,
would you send me back the PDF format of this paper?
many thanks.
I guess you want to synthesis cDNA of mRNA. In that case, you have to use reverse primer, only.
Be careful your RNA does not have a modified base in it. Some modifications slow RT (dimethyl G), others are absolute stops (am psi). Some have no effect (pseudouracil). My Ph.D. thesis was on this topic, published in 1981 with John E. Hearst at UC Berkeley. Chapters were published in PNAS and Nucleic Acid Research.
What is the purpose of your cDNA synthesis? Are you going after one gene to clone and sequence or protein expression study? Then GSP will work. It also works on low abundant transcripts for the above purpose. Are you trying to do QPCR? For QPCR it is better to use Random Hexamer primers. Because for RT QPCR you need to amplify your gene of interest and some house keeping gene to normalize expression. Like Dr. Rocco Piazza mentioned if you use gene specific primers you won't be able to amplify other genes. Hope this helps. Best wishes.
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