Hi
I have run into trouble. I'm working on the gene expression measuring, but it hasn’t successful finally. I did check and replace all steps in experiments but have still not concluded, also Realtime PCR is my method.
I’m using RNA extraction kits made by Roch companies(Tripure)
DNAse for DNAse Treatment made by fermentas(Thermo Scientific).
Geneall cDNA synthesis master mix
And Evagreen mastermix made by Biodyne for Realtime PCR.
I did change all procedures many times. For example for washing RNA have used at first ethanol (70%) then 96% in another specimen. I did change primers several times, with a length of 76, 94, 150 and 200 bp. Times and temperature cycles of Realtime PCR have changed frequently. Realtime PCR device from Qiagen and ABI have used, but the experiments failed again. On the other hand more samples in PCR made band on the gel electrophoresis but result of Realtime PCR is unsuccessful. My specimens are extracted from animal tissue.
Please guide me.
Grateful
There are two things to check first.
1. Check your RNA band after RNA extraction to make sure the RNA has been extracted.
2. Exam the RNA concentration. When I do the RT-PCR, I added about 1ug RNA to get cDNA, and the result is good.
Good luck with you
There are two things to check first.
1. Check your RNA band after RNA extraction to make sure the RNA has been extracted.
2. Exam the RNA concentration. When I do the RT-PCR, I added about 1ug RNA to get cDNA, and the result is good.
Good luck with you
You may want to check your RNA integrity first with agarose gel or bioanalyzer.
Sometimes the respective transcripts are low abundant in your sample tissues. So you need to have high concentration or amount of RNA.
Apart from that, you have to make sure you get high quality of RNA by checking them using agarose gel and bioanalyzer.
You may also refer this link for further information and guidelines.
http://miqe.gene-quantification.info/
Hi Ali,
As suggested above by researchers,,you need to do it carefully...and always keep a control reaction first to see if the things are working fine....assess quality of the template (RNA, cDNA etc.) at each step of the protocol....there must be some minor problem somewhere....check carefuly
Dear Ali,
When I read DNase treatment, I remember one case in which DNase was not 100% inactivated before PCR. DNase zap is a kit to efficiently inactivate DNase. If not, your primers and cDNA will be digested.
First, you can test this by adding a plasmidic DNA as a template of pcr to one of your rt reactions, at a low quantity, and then perform the qpcr.
I hope you got the idea...
Puri
Dear Ali
All the above research have explain everything in details. i Have another view points. What you use for the lysis of the cells. Firstly check that your lysis reagents is working on the specified tissue or cells. Secondly after extraction of the mRNA can you see the pellet visually ?. But most probably you need to change your lysis reagent
Dear Ali,
All the above enswers are right and are usually the first things to test, however, you said that in semi-quantitative PCR you have band, but in Real Time PCR (qPCR) you don't. Am I right?
For me the problem is the compatibility between DNAse and qPCR kit. There are qPCR mixtures that have low compatibility with DNAse synthesis kit.
I would suggest that you use both kit from the same brand.
Best of luck
Dear Ali,
I would suggest designing shorter primers 20-30 bp and check them first with a semi-quatitative PCR.
Good luck
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