I have isolated plant RNA from Qiagen kit. The 260/280 and 260/230 ratio are all above 2, the RNA looks good on the gel, then when I make cDNA and do real-time, 2 out of 6 samples show nothing on real-time for 5 genes tested, even for the endogenous control EF1a. 2 out of the remaining 4 are not so good, while the last 2 are the best. The RT PCR mix was same for all 6 samples and so was the real-time reaction mix. The cDNA yield looks good on nanodrop for all. What could be the reason that cDNA is working bad for some samples?
Hi,
May be you miss RNA prior to cDNA synthesize;
The relevant gene my not express in your sample;
Have you got good results on reference gene?
Best
I cannot forget to add RNA in 4 samples, thats not possible. For 2 of them even the reference gene didn't work. it has become a mystery for me now.
There are several possibilities:
Check the integrity of the RNA by denaturing agarose gel electrophoresis
RNA should have a minimum A260/A280 ratio of 1.7 or higher. Ethanol precipitation followed by a 70% ethanol wash can remove most contaminants such as EDTA and guanidinium. Precipitation with lithium chloride can remove polysaccharides
Phenol/chloroform extraction and ethanol extraction can remove contaminant proteins such as proteases
Some target RNA may contain strong pauses for RT; Use random priming instead of d(T)23VN.
Use sufficient amount of RNA.
Hi
1)Though RNA gave you good ratio you need to check its integrity by running on the gel.
2) Your 2 samples failed even house keeping amplification that means your RNA may not be amplifiable.
3) Re purify your RNA.
4)Do you use rendom hexamer or oligodT? Some times mixing both helps.
Shefali
Hi for every one
one step RT qPCR is more effective than 2step, so you can use your RNA samples directly and don't forget using +ve and -ve control.
Good Luck
Zaid, if Sachin's RNA isn't good neither one nor two step will work. I believe his samples have some inhibitor carry over, so one step won't correct the problem.
Thanks to all of you for your replies. What inhibitor carry over could be, I am using the Qiagen kit to clean it up, one wash with RWT buffer and then two washes with RPE buffer and then eluting with water. And I used the same treatment for all samples. Two sample came out very good. I take the plant with roots intact from the medium and then I remove the medium attached to the roots by kim wipe and dry i a little bit and then crush the tissues. With kit all inhibitors should be removed and RNA on agarose gel looks very intact, no degradation.
Sachin before we can say for sure you need to be sure your primers are good and you need to perform an inhibition curve. Take different concentrations of RNA and perform RT followed by qPCR. You should see a straigt line (Ct vs [RNA]). If you get a plateau in the samples with higher RNA concentration than you have the presence of a inhibitor.
If the inhibitor is confirmed I believe the major culprit are polyssacharides that are abundant in nucleic acids extracted from plants. You could try treating your RNAs with RNAse free hydrolase and than repurify your RNA.
Before you do that did you check your primer sequence and if they work indeed?
Hi Andre, thanks again. yes, primers are fine, I am using the same set of primers in the same reaction master mix for other samples and they are working well. Only for some samples none of the primers are working, indicating the cDNA is not there, although the cDNA concentration on the nanodrop is the same as the samples which are working.
Then titrating your RNA before RT to assess for the presence of inhibitors can be a solution. If you find a RNA dilution that all samples work than your problem is practically solved. Take a look at the application note bellow it may help.
Is your Qiagen kit suitable for plants? Since this kind of sample contains a lot of polyssacharides you may need to add a high concentration of salt to avoid that polyssacharides precipitate with the RNA.
http://www.nanodrop.com/Library/T111-Detection-and-Avoidance-of-Polysaccharides-in-Plant-Nucleic-Acid-Extractions.pdf
http://www.jstor.org/stable/1223408?seq=1#page_scan_tab_contents
I think you are right, now I recall, first time when I used less RNA I got my housekeeping gene in the real-time, but it gave inconsistent Ct values, I thought the RNA might be less, so second time I took more input RNA to make cDNA and then even the housekeeping gene failed, a flat line in the Ct curve, no plateau. The other genes never worked. So you are correct I think something inhibitory in the RNA and these polysaccharides are coming from the roots, I believe. I first extract by trizol and then pass through miRNeasy column of Qiagen and wash there. After trizol extraction steps are similar to animal extraction, but yes, I will ask the company if I have to wash with the plant kit specific buffer, instead of RWT in this kit. In the plant kit we pass through Qiashredder column first to filter out the crushed debris, which in this approach I do by centrifugation and then taking the supernatant. We extract RNA by manual method this way, so I am just using the kit column to get cleaner RNA. I will talk to Qiagen people. And these plants are little older so they should have more polysaccharides, this problem was more seen in older plants.
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