I am using Taqman miRNA assay kit for miRNA amplification. But I am getting the peak for water control also. I have repeated by using new purchased RNAse free water, even-though I am getting same problem.Please suggest me how can I resolve my problem?
It is hard to say exactly since there is no data on what kind of peak you are getting. Does the peak in your negative control cross your CT threshold? Does it appear to follow a sigmoidal curve or is it linear? A lot of that information can help to ascertain where in the process you are getting contamination. Also are you working in a separate area from other areas where you perform PCR or work on any downstream applications? Amplicon can become a big issue if you are using the same hood to set up RT-PCR as you are purifying PCR product.
Dear Poolandanda,
Your kit is contaminated with RNA so you see the expression of it in both control and samples. You should use another kit.
Sincerely,
Sercan
thank you sir,
I have tried with new kit also, but same problem i am facing.
And sir, i am using taqman small RNA assay 20X 1 um
RT product 1.33 um
SYBR green master mix 2X 10 um
Nuclease free water 7.67 um
total 20 ul
experimental conditions: preincubation 95 degree centigrade for min
denaturation 95 degree centi for 15 sec and annealing 60 degree centi
melting and cooling temp and time as per Light cycler 2.0 i have proceeded.
And I am requested to Arthur sir, I am not that much experienced with this technique. I have tried with all suggestions from my guide even-though i am getting this problem. And also I know how to use RG. Please encourage us.
Since you have replaced kit and water I would recommend that you make sure your working area is not contaminated with amplicon. If you are preparing your reaction in the same hood that you are also using or have used product then there is a high chance you have gotten PCR amplicon in your new reactions. If you used it the first time without contamination and have been experiencing contamination with every subsequent run then I would check your work space. You can do a swipe test from your bench area, also run an entirely negative control as well as various combinations of kit.
I think since you mentioned "Taqman" in your original question, people here (including me) most likely assumed you were using a hydrolysis probe-based qPCR method. Now that you've stated that you are using a SYBR Green master mix, you could merely be experiencing a primer dimer issue here. Try 62C annealing temperature and/or reduce amount of primers added, and you may be able to improve your results. Trial and error on this should help. Good Luck~
Also see the RG post:
https://www.researchgate.net/post/Why_do_I_get_small_bands_primer_dimer_in_my_PCR?tpr_view=a0552551-4799-4b99-b426-b6aeccc6bac9_2
thank you sir for your kind information. I can proceed yours suggestion.
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