The cDNA I have synthesized was from a mixture of mRNA from fungus and plant tissues (as I'm studying interactions). I have a problem in getting the required quantity of cDNA which I can readily amplify in RT-PCR (being too low). As a result the exponential amplification of my RT-PCR started after 35 Tc. Still the standared curve appeared to be not good. The red spots are not coinciding the straight standard curve (line) indicating bad results. So how would I improve the quality and quantity of my cDNA so that there shall be good standard curve?
are you sure your RNA is of sufficient quality?
If you have degraded RNA, it's no surprise you get very high Ct's.
I don't know which cDNA synthesis protocol you used, but if your using oligoDt (poly-T primers, which target the poly-A tail of the mRNA) you'l only amplify intact mRNA, starting from the poly-A tail. In case of degraded RNA you could increase your success rates by adding some random primers (random hexamers) to your cDNA protocol, to also start cDNA synthesis along the length of your RNA.
And ofcource imperative, try designing your qPCR assays as short as possible, and intron spanning. Mine are nearly always below 150bp, which increase PCR efficiency, and also is less susceptible to problems with differences in RNA quality.
You should check, both spectrophotometrically and by agarose gel electrophoresis, if your mRNA and/or cDNA is of good quality.
Agarose gel electrophoresis will show you the amount of your RNA and whether it is degraded or not. Spectrophotometrical quantification will also show you the quality of the isolated RNA.
@Arnoud Boot, The quality of my RNA was good as long as there were two bands revealed by agarose gel. The protocol am using to synthesis cDNA is IM-PROM-II reverse transcription system kit. I'm doing the quantification in two steps...cDNA synthesis and then RT-PCR which uses oligo dT primer. The primers am using are designed based on ESTs not based on geneomic DNA and hence no worries about intron spanning. The only thing I am thinking to do is to use random primers as you said. I wonder if some left overs of cDNA synthesis (primer, DNTs,,,) could have an impact on RT-PCR amplifications.
Another recommendation for a good synthesis of cDNA, it is better use a RNA very concentrated (if it is possible 1000 or 2000 ng/uL), because sometimes during the extractions you could drag undesirable elements that could inhibit cDNA synthesis. Thus you have to take just a little volume of solution and you´ll have just a little concentration of this elements.
make sure your RNA quality, then DNase I treatment , then try the super transcriptase III from invitrogen
What do you use to make the standard curve? A serial dilution of one sample? A mixture of samples? plasmid?
The way your standard dilution looks tells a lot about you pipetting skills/experience. Maybe try to have someone with more pipetting experience make your serial dilution and RT-qPCR reaction mixtures. At what Ct does the first point in your serial dilution come up?
To test if your cDNA is allright, you could try to make a standard curve with other primers (e.g. a reference gene)
@Britt, txs for the sugestions. I'm doing a serial dilution of one sample to make the standard curve. Of course am a beginner, hence there could be errors during pipetting though I steadly improve in the course of my trial. Ok I will try to make standard curve using reference gene in my net run. Txs again.
Dear Gelata first check your RNA sample purity I think your RNA may not be good so that you couldn't get good curve so revive the procedure
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques