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Low amount of RNA and cDNa for qPCR


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#1 katiiiiii

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Posted 04 February 2019 - 01:23 AM

Dear all,

 

I have a big problem. I used the wrong RNA extraction kit for my sorted B-cells (around 500 000 cells).

This results in a low RNA concentration in my samples (around 5ng/µL measured by nanodrop - i know it is not a precise measure).

I did my cDNA synthesis with two different kits (RevertAid First Strand cDNA Synthesis Kit and High-Capacity cDNA Reverse Transcription Kit - Applied Biosystems) and I can't detect signals in qPCR experiment.

 

Do you know a good kit for cDNA synthesis for low amount of RNA or a method to concentrate my rna or cDNA?

 

Best, Kati



#2 OldCloner

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Posted 04 February 2019 - 06:07 AM

First, Kati, Exactly how wrong was the kit you used for RNA extraction? What was it designed for and what were you trying to get (total RNA, mRNA, etc.)? Also, are B-cells very active transcriptionally? If not you might not expect a lot of mRNA.

 

You would benefit from a way to check the quality of the RNA you extracted. Nanodrop tells you nothing about RNA integrity.  If you have access to a lab with an Agilent Bioanalyzer (which uses chip technology), or some other equivalent technology, you can see if it is good stuff (proper ratio of ribosomal RNA peaks) or just degraded ribonucleotides.  There are chips for low concentration RNAs. Or you can run an old-fashioned RNA gel to assess the same.  If the RNA is badly degraded no amount of concentration will help. If these options are not available, (or too expensive) try a PCR with some high-success primers (a strongly expressed control gene for your cell type) and if that doesn’t work, it is probably best to write this one off and start over.

 

You can concentrate nucleic acids by old-fashioned ethanol precipitation with salts: look up the methods in a lab manual (or on line) as the pH of the salts used differ for RNA vs. DNA. But if you have very low amounts you might be disappointed with the final conc. as some will inevitably be lost. If it is degraded you will lose all of it.

 

I typically used Qiagen kits for nucleic acid extractions, and Invitrogen Superscript III kits (now under ThermoFisher) for my RT reactions, and was generally happy with them. You have to consider the type of oligo to use when you are reverse-transcribing (oligo-dT or random) as that influences the amount of cDNA product you will get. For maximum output you can mix both types of oligos, in fact some kits come with mixed oligos.

 

Good luck!


Edited by OldCloner, 04 February 2019 - 10:44 AM.





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