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Normalization for RNA or cDNA during two step RT-PCR?


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17 replies to this topic

#16 hasheminia

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Posted 28 June 2010 - 09:14 AM

hi-do you know qiashredder homogenizer is a serious need for rna extraction from macrophage or not? ihave 100000 cells and need about 100 ng rna .
thanks :hasheminia

#17 Harvey

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Posted 13 March 2011 - 07:08 AM

You'd better normalize against total RNA prior reverse transcription.

Sure, cDNA clean-up enhances the PCR. In most cases, we didn't purify cDNA. Then limited volume of cDNA should be added to a PCR reaction

#18 Nephrite

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Posted 28 March 2011 - 03:14 AM

Hello :-)

Excuse my stupid questions.

About this RNA normalization..... Here I was given an RNA quality control protocol, you can see it.

1. What is this RNA normalization?

2. How someone could normalize the amount of mRNA if it not visible on the gel, as seen in this protocol?

3. Apart from how intact is my RNA, what more information gives me the RNA electrophoresis, if I see only the rRNA?

4. If I load my RNA sample on a 1% agarose and I don`t see anything on the level of the upper marker, would it mean that the sample is not being contaminated with gDNA?

5. As far as I undestand, non-PCR-ed gDNA makes something like.....bands with approximate size at about.....8000-10 000 bp, is that correct?

Any answer will be highly appreciated,

Nephrite

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