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RT-PCR Negative controls


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#1 Epic

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Posted 14 April 2009 - 08:00 AM

Hello!!

Can someone define the use of -NE controls during PCR? What do you use for negatibe controls?
As negative controls I use 2:
RNA -ve (contains all cDNA components but not sample RNA)
RT-ve (contains all PCR components but not sample cDNA)

Cheers

#2 almost a doctor

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Posted 14 April 2009 - 08:29 AM

Hello!!

Can someone define the use of -NE controls during PCR? What do you use for negatibe controls?
As negative controls I use 2:
RNA -ve (contains all cDNA components but not sample RNA)
RT-ve (contains all PCR components but not sample cDNA)

Cheers



Hello, I usually have 2 negative controls. They are slightly different from yours.

RT -ve control: this for me is actually all the RT reagents + RNA but NO RT enzyme. This way you have a control for presence of gDNA, and also show that on your RT +ve the reaction is happening.

NTC (no template control): this has all the PCR reagents but no DNA/cDNA (template). This will show that your PCR is specific and free of contamination.

If your RT -ve shows up in PCR: you have DNA in your RNA sample.

hope this helps ;)




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