Has anyone worked Real Time PCR with cDNA previously transcribed from RNA that had a low 260/230 ratio, around 1? How does this affect the gene expression in Real Time PCR analysis ?
We actually never checked our cDNA preps, we only diluted them and used them right away. Do you purify your cDNA after the prep? If not, you still have proteins (the reverse transcriptase) and buffer components from your cDNA reaction present, which will affect the OD measurement. The pH also influences your measurement, acidic pH lowers the ratio by 0,2-0,3, basic pH does the same thing in the other direction.
I suggest that you run regular PCR on the cDNA with primers designed to amplify housekeeping genes. Based on the results you can see more or less tell whether your cDNA is viable.
Hope this helps! Best of luck!
Christian, I don't purify cDNA, just check concentration on NanoDrop. Through 260/280 ratio i see if i have protein contamination (260/280>1.8 biblical value of PCR). Alicia, yes I run regular real time pcr and indeed my idea was that through efficiency of pcr amplification of housekeeping gene I can see whether my cDNA is viable? You agree?:-)
I agree! Unless there're some other ways of going about it that I've yet to hear about.
Best of luck Milos :)
To make it clearer, till now I was doing isolation from rat brain tissue and I had no problem nor with isolation, reverse transcription or pcr. Now, I am optimizing protocol with human blood lymphocytes. During that, I had a problem with isolation of RNA, I have a small rna concentration (from 3 ml of whole blood - isolation of lymphocytes with ficoll - isolation of rna using trizol - rna concentration in the range 150-200 ng/ul or approximately 3 ug of total amount of rna). Also, a 260/230 ratio was very small, about 1, so i am wondering if this is going to be a problem during pcr?? Also, this small ratio is probably a result not of contamination of rna during isolation, whereas to small absorbance on 260 which is result of small rna concentration (also had a small 260/280 ratio that confirms low concentration of rna).
The NanoDrop is very useful for higher concentration measurements, but it doesn't tell you much about the integrity of your sample, and you don't necessarily have to eliminate samples based on 260/230 ratio. Yes, you want to check efficiency at the PCR step, as you said. You might also want to check the reverse transcription step by spiking a known RNA into your reaction mix (something not found in human, in your case) and measuring this for all samples along with your target(s) of interest and internal control(s). This way, you can assess whether RT or PCR was inhibited by something contributing to the low 260/230 ratio.
I would suggest to use Agilent Bioanalyzer to check for the quantity and integrity of the RNA. It is likely that you may have poor quality of the RNA that may lead to the poor reverse transcription. As other suggested, there could be other reasons and including a internal spiking control will also helpful to understand the problem.
Kenneth can you explain me the "spiking a known RNA into reaction mix? I understand but not in complete.
Just go for a gel check and see if your RNA is intact, that u see all the bands clearly. Then dilute it and run a routine PCR for housekeeping gene. It should work...
Milos, just as you might add a spike-in control at the purification step to assess RNA recovery, you can also add a spike-in control to the reverse transcription reaction. Measuring the spike-in along with the targets and normalizers would identify problems with reverse transcription.
The integrity depends also on the amount of RNA. So you must have enough quantity (>5μg). The electrophoresis of qPCR products will tell you the "true". The most cDNA kits amplify almost everything, even if you use oligodTs. The expected PCR products will help you to reject the non-specific bands.
If you are interested only in a few target genes (as you probably are if you are planning on doing real-time PCR), an alternative to the use of RNA spike-ins would be to run serial dilutions of your "suspicious" samples with your target and reference primers in real-time PCR. You will then see if your samples contamination or degradation will interfere with your amplification: if your amplification efficiency is still good (close to 100%) and the melting temperature the same as expected (or the same as positive control samples), then you could be confident in your samples.
The effects of your contamination can appear more or less important depending on the levels of expression of your target genes, so this could help you estimate those effects.
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