I have set of 7 tissues each for treatment and control groups. RNA integrity and concentration for all 14 tissues is good. Further, I treated with DNase 1 prior to cDNA synthesis. We use RevertAid first strand cDNA synthesis Kit for the same. The similar conc. of the template (cDNA) was added for qRT-PCR reactions. But on analyzing qRTpCR result I found different Ct values for even b-actin which ranged from almost 12-20 in case of all 14 tissues. Can anyone suggest me ways to solve this problem because
Actually we measure the RNA concentration by nanodrop through which we take 1ug of RNA, treat with DNAse 1 and perform cDNA synthesis for 20uL. Then I added 1uL of cDNA for 10 uL of qRTPCR reaction (each reaction), that means similar concentrations of cDNA as well, then why this problem.
Yeah, and I think may be other positive control candidates can be a good option, but still is there no solution for b-actin as endogenous control. Thanks anyway.
Hi Bhakti, please refer the following link.
http://www.protocol-online.org/biology-forums/posts/24068.html
Hi, have you consider that if you are analyzing different tissues b-actin may have different expression levels and, therefore, not be the best reference/housekeeping gene. Other possibility is that you are adding to much cDNA in that 1ul cDNA, have you tried serial dilutions? The link provided by Badrinath Narayanasamy has very useful tips, please check it as well.
good luck
Hi guys,
Sorry Bhakti to add question to urs here. but by reading Jose's answer, one query came to my mind. I have tried all the controls mentioned B-actin, GAPDH, B2M and Rn and they are good in lower concentrations of my treatment 10, 20 and 50mM but when I increased the concentration to 100, the internal control Ct increased dramatically up to 25? Do u guys have any explanation plz knowing that the other genes remain almost the same as with 50, 20mM of the treatment?
Thanks
Thanks Alfonso, but I have tried with 3 cDNA dilutions, 2X, 3X and 5X but in 3X i was getting the best melting peak hence, I chose that dilution for my entire experiment, and I want to add regarding the pipetting error, that I have run triplicates as well, but they are exactly similar.
If you are confident you are adding the same amount of cDNA then beta-actin may simply not be a very good housekeeping gene for your experimental paradigm. I would validate a number of HKGs and use genorm to find the most stable.
I completely agree with Alison Brain. I would only add that it is a good idea to have negative controls (H2O instead of cDNA) to be sure that you do not have any contaminants messing with your reactions.
good luck!
It happened the same to me, I am using B-actin as HK for liver and spleen tissue but the values of the replicates differ from each other, and the negative control resulted negative.
I am also facing the same problem as Bhakti is facing. I am also using beta-actin as a positive control for gastric carcinoma tissue. I have tried 7 times and every time the Ct value is different. my system is of Bio-Rad and the syber master mix don't have ROX solution. it has been provided separately. but in the kit information, it's clearly written about the compatibility of the kit with different systems. and my real-time machine doesn't require ROX solution. but my mind is thinking like it may be coz of ROX solution. what shall I do plz suggest me help me.
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