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Reverse transcription (cDNA synthesis) curiosity and confusion


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#1 kedar

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Posted 05 May 2011 - 04:05 AM

Dear all,

Here is my protocol.
1 microgm mRNA (taken to the volume of 10 micolt with H20)
1 microlt randomprimers (500 ng/microlt)
1 microlt dNTPs mix (1 mM)

incubate 5 min 65 degree.

4 micrlt 5x FS buffer
2 microlt 0.1 DTT
1 microlt RNASE out
1 microlt reverse transcriptase

Incubate 10 min 25 deg, 59 mn 42 deg and 15 min 70 deg.
final volume - 20 microlt.

NOW MY QUESTION IS: tO PREPARE MORE Cdna I.E. 40 MICROLT INSTEAD OF 20 MICROLT, I DOUBLE EVERYTHING BUT NOT THE mRNA....because my view is the same template of mRNA can be used again and again by the enzyme.
apparently, when i run the realtime pcr, it's not working..it's getting amplified at 33 or more cycles, with no template control showing the same amplication as samples where cDNA was added.

Does this mean, if i want to prepare more cDNA, i also have to use double (2 microgm) mRNA while doing the RT.


your advice will be appreciated.

many thanks

#2 Trof

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Posted 05 May 2011 - 04:53 AM

Does this mean, if i want to prepare more cDNA, i also have to use double (2 microgm) mRNA while doing the RT.

Yes. Reverse transcription is not cycling, the template is transcribed only once. Actually you don't need to double anything but the amount of RNA, if your RT mix is capable to transcribe more template (check the manual).
For example Roche Transcriptor I use can transcribe from 10ng - 5 ug, I usually put 1 ug to the reaction, but when I need bigger volume, I transcribe 2 ug and then dilute it to 40 ul at the same cost. If I needed more concentrated cDNA I wouldn't dilute it.

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#3 kedar

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Posted 05 May 2011 - 05:01 AM


Does this mean, if i want to prepare more cDNA, i also have to use double (2 microgm) mRNA while doing the RT.

Yes. Reverse transcription is not cycling, the template is transcribed only once. Actually you don't need to double anything but the amount of RNA, if your RT mix is capable to transcribe more template (check the manual).
For example Roche Transcriptor I use can transcribe from 10ng - 5 ug, I usually put 1 ug to the reaction, but when I need bigger volume, I transcribe 2 ug and then dilute it to 40 ul at the same cost. If I needed more concentrated cDNA I wouldn't dilute it.


thanks Trof. got it.

I transcribe 2 ug and then dilute it to 40 ul at the same cost.-------- same cost, you mean, for e.g.- to make it 2 microgm in 40 microlt, let's say i need 15 microlt of my mRNA, then i still use random primers, dntps etc in the same amount as for 20 microlt??? and the rest is water...
do i understand this right?

Edited by kedar, 05 May 2011 - 05:02 AM.


#4 kedar

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Posted 05 May 2011 - 05:27 AM

Hi nephrit,

not really mRNA, it's whole rna.
yea, but 1 microgm is not much too..many labs use this amount for RT. but i don't deny the fact that it might inhibit the template.
no, cDNA cannot be measured i.e. why you should know the amount of RNA that you use for RT, meaning that you are assuming that you are using 1 microgm rna for real-time pcr in every well.

Edited by kedar, 05 May 2011 - 05:28 AM.


#5 Trof

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Posted 05 May 2011 - 07:32 AM

Well, I usually get more concentrated RNA (300-1000 ng/ul) so I put there 2 ug right from the stock to the same reaction I would put 1 ug, that means 20ul reaction (and fill up with water to get the right volume). Everything stays the same, there is just 2x more RNA. After the RT is finished, I dilute the cDNA to have more uls for more PCR reactions. But you must check what template range is your RT suitable for.
If you have less concentrated RNA though, you are limited by the maximum volume you can add to the reaction (in my case with Transcriptor it's 10 ul, but if my RNA is say 300 ng/ul that's 6.7 ul for 2 ug and rest is water; in your case the maximum is 10 ul too).

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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