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


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#1 Curtis

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Posted 20 April 2009 - 02:13 AM

Hello all,

I have a two-step SYBR Green RT-PCR kit for RotorGene. After I extract RNA from my samples, I proceed with cDNA synthesis right away without measuring the RNA concentration or without normalizing equal amount of RNA for all my samples. It is only after making cDNA that I measure the concentration and add around 100 ng in each PCR tube.

my results are a bit funny now and I get my first signal after 35 cycles and surprisingly the samples that should give signal sooner are always the last ones. my friends are suggesting to measure my RNA before proceeding with cDNA synthesis. They say maybe too much RNA in the sample does not let cDNA to be properly synthesized !?

is this comment true?

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#2 littleaxt

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Posted 20 April 2009 - 04:42 AM

Hi

How do you measure cDNA concentration? Do you purify it before measureing it? Because I was told that if I measure with the Nanodrop I would also measure the unused dNTPs and therefore I could also start making it up :-)...


Hello all,

I have a two-step SYBR Green RT-PCR kit for RotorGene. After I extract RNA from my samples, I proceed with cDNA synthesis right away without measuring the RNA concentration or without normalizing equal amount of RNA for all my samples. It is only after making cDNA that I measure the concentration and add around 100 ng in each PCR tube.

my results are a bit funny now and I get my first signal after 35 cycles and surprisingly the samples that should give signal sooner are always the last ones. my friends are suggesting to measure my RNA before proceeding with cDNA synthesis. They say maybe too much RNA in the sample does not let cDNA to be properly synthesized !?

is this comment true?



#3 Curtis

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Posted 20 April 2009 - 05:45 AM

good point, but if I purify the cDNa I'm gonna lose a lot of it during the purification steps. after all we are not talking about a PCR product that has been amplified. it's just cDNA from our RNA which is also not a lot.

#4 gfischer

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Posted 20 April 2009 - 06:45 AM

I always normalize the amount of RNA I use in my RT reaction. This way, you know that the only source of variation in the amount of signal in the quantitative step is from a higher level of relative expression of target RNA.
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#5 littleaxt

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Posted 20 April 2009 - 07:48 AM

Oh, it seems possible, see http://www.biomedcen.../1471-2199/9/18 they also did it. Seems like cDNA clean-up also enhances the PCR.

But I never tried it myself, I normalize against total RNA prior reverse transcription and use relative quantification.

All the best

Jan

good point, but if I purify the cDNa I'm gonna lose a lot of it during the purification steps. after all we are not talking about a PCR product that has been amplified. it's just cDNA from our RNA which is also not a lot.



#6 Curtis

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Posted 20 April 2009 - 08:07 AM

littleaxt: yeah, relative quantification. that's what I'm doing now. the link you gave doesn't open for me.

gfischer: that would be for one-step RT-PCR? or applies to two-step as well? how do you make sure you won't have more than 100 ng of cDNA in your sample? because if you have more than 100 ng the amplification might not work properly.

#7 littleaxt

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Posted 20 April 2009 - 11:04 PM

ok, this might help:

Libus & Štorchová 2006 “Quantification of cDNA generated by reverse transcription of total RNA provides a simple alternative tool for quantitative RT-PCR normalization” BioTechniques 41, 156-164

Liss 2002 “Improved quantitative real-time RT-PCR for expression profiling of individual cells” Nucleic Acids Research 30 (17): e89

Hibbeler et al. 2008 “Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus“ BMC Molecular Biology 8:18

First is another idea you could try, second is a precipitation protocol for cDNA (never tried it myself) third is another paper where they made a cDNA clean-up prior PCR. Hope it helps, good luck!


littleaxt: yeah, relative quantification. that's what I'm doing now. the link you gave doesn't open for me.

gfischer: that would be for one-step RT-PCR? or applies to two-step as well? how do you make sure you won't have more than 100 ng of cDNA in your sample? because if you have more than 100 ng the amplification might not work properly.



#8 Curtis

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Posted 21 April 2009 - 06:08 AM

ok thank you, I'll have a look now

#9 Trof

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Posted 22 April 2009 - 03:33 AM

We do two-step RT real-time PCR and always measure the amount of RNA only. Usually we put 1-3 ug of RNA to the RT reaction, depending how much cDNA we need. We don't measure nor clean-up the cDNA, use it directly to the real-time PCR.
We can dilute the cDNA if we need more volume for more samples (like 2 times or 3 times), but usually only when using more than 1 ug. Never exceed the 10 % of cDNA in a reaction mix. That makes from 1 ug in 20ul RT mix, when using maximum 2ul to a 20ul PCR reaction just about 100 ng.

I think we would see it on the aplification curve if there is any problem with high amount of cDNA, we never encountered such thing.

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#10 Curtis

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Posted 23 April 2009 - 09:18 PM

We do two-step RT real-time PCR and always measure the amount of RNA only. Usually we put 1-3 ug of RNA to the RT reaction, depending how much cDNA we need. We don't measure nor clean-up the cDNA, use it directly to the real-time PCR.
We can dilute the cDNA if we need more volume for more samples (like 2 times or 3 times), but usually only when using more than 1 ug. Never exceed the 10 % of cDNA in a reaction mix. That makes from 1 ug in 20ul RT mix, when using maximum 2ul to a 20ul PCR reaction just about 100 ng.

I think we would see it on the aplification curve if there is any problem with high amount of cDNA, we never encountered such thing.


Thank you Trof,
your explanation was very helpful.

#11 Chimp

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Posted 15 June 2009 - 11:54 PM

Checking [RNA] is an important step. You could also run a denaturing RNA gel to check RNA integrity, you should get clear rRNA bands

#12 Freddynb

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Posted 02 September 2009 - 04:43 AM

I have developed a method to amplify multiple random genes in a single real-time RT-PCR reaction to assess quality of cDNA between samples. It can be used to normalize gene expression between samples. Please see
http://www.springerl...28327382188710/

#13 sssbio

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Posted 29 May 2010 - 02:32 PM

I have developed a method to amplify multiple random genes in a single real-time RT-PCR reaction to assess quality of cDNA between samples. It can be used to normalize gene expression between samples. Please see
http://www.springerl...28327382188710/


Hi,

I need your help regarding quantification of mRNA levels of trageted genes in control and treated samples. I have no choice of reference housekeeping genes to be used for relative quantification as these genes are showing significant down regulation in treated samples. So what are other methods that I can use to quantify mRNA levels?

I will be very happy if you guide me for this.

Thanks

#14 gradechips

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Posted 02 June 2010 - 10:05 PM

Please help me, I have been doing RT-PCR for the past six months and i still have problems. During this period i was only able to get decent Cts for the house keeping gene (18) twice but all the other times the Cts for both GAPDH/beta actin have been really high 25 or inconsistent within triplicate samples (20, 24, 29). Obviously i cant use these data to analyze gene expression and am getting frustrated. After extracting RNA using Trizol i quantify it by ODing and i usually get ~2.500ng/ul and i use 2ug for the cDNA synthesis and i normally dilute the cDNA 1:50. My lab mate normally gets low and consistent Ct for the house keeping gene using the same reagents. I have been trouble shooting for the past 6 months with his help but am not getting it.
Is it because vortexing shear my samples? Please someone help me.
Thanks in advance

#15 Nataliya

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Posted 24 June 2010 - 12:19 AM

Please help me, I have been doing RT-PCR for the past six months and i still have problems. During this period i was only able to get decent Cts for the house keeping gene (18) twice but all the other times the Cts for both GAPDH/beta actin have been really high 25 or inconsistent within triplicate samples (20, 24, 29). Obviously i cant use these data to analyze gene expression and am getting frustrated. After extracting RNA using Trizol i quantify it by ODing and i usually get ~2.500ng/ul and i use 2ug for the cDNA synthesis and i normally dilute the cDNA 1:50. My lab mate normally gets low and consistent Ct for the house keeping gene using the same reagents. I have been trouble shooting for the past 6 months with his help but am not getting it.
Is it because vortexing shear my samples? Please someone help me.
Thanks in advance



I usually use RNeasy Mini Kit (50) and RNase-Free DNase Set (50) from Qiagen. Once I have got RNA extracted I use it for cDNA synthesis or keep at -70 C. For cDNA synthesis I used SuperScriptTM III First-Strand Synthesis System for RT-PCR (Invitrogen, Cat. No: 18080-051). For RT-PCR I diluted cDNA 1:5. Sometimes I measured cDNA by nanodrop and the concentration of the diluted cDNA samples was around 200 ng/ul I have never faced the problem with low values for house-keeping genes. I normally get 13-14 Ct for GAPDH. Probably, you don't need to dilute samples up to 50 ul/ml.




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