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Totai RNA dilution - (Aug/02/2011 )

Hi,everyone
I am doing microRNA relative quantification using taq-man microRNA assay.
I have isolated total RNA from Huh7-mock and Huh7-HCV replicon, and the concentration is 498ng/microliter and 520ng/microliter,respectively.
I have diluted total RNA to 10ng/microliter, and run RT and qPCR.However,the relative expression of miR-2XX is no difference between Huh7-mock and Huh7-HCV replicon (deltadeltaCt=1.22).
My question is that whether the dilution of total RNA affects the quantification of microRNA or not.
Should I use no-diluted total RNA for RT-qPCR?
ps. the relative expression of miR-2XX between Huh7-mock and Huh7-HCV replicon is more than 2-fold.

-yueh76-

What is the difference between these two cell lines--one expresses the miRNA?

It should not matter much whether you dilute your RNA or not as long as you use the same amount of RNA for your RT reaction. However, the amount of RNA should be in a reasonable range. It seems that the amount of RNA you used is very low and the RNA was too diluted. Can you tell use the exact amount of RNA you used in the RT reaction?

-pcrman-

According to Taqman protocol, the amount of RNA is 10ng in each 15ul RT reaction. If you use non-diluted RNA to run qPCR, Ct will become small. Especially for internal control, sometimes its Ct will be lower than 20. That means too much template is in each reaction. If Ct of your target is around 25, I don't think it is necessary to increase the amount of template. Even you increase RNA, it will not change the relative expression. Actually, I think the efficiency of PCR and the quality of cDNA are two major factors that affect the reproducibility.
By the way, why do you ensure the relative expression should be over 2-fold? Do you get this information from other paper or microarray? If it is from microarray, maybe it is not 100% reliable.

-txyuan-

pcrman on Tue Aug 23 01:56:27 2011 said:


What is the difference between these two cell lines--one expresses the miRNA?

It should not matter much whether you dilute your RNA or not as long as you use the same amount of RNA for your RT reaction. However, the amount of RNA should be in a reasonable range. It seems that the amount of RNA you used is very low and the RNA was too diluted. Can you tell use the exact amount of RNA you used in the RT reaction?

I used 10ng total RNA for miRNA RT reaction (according to Taqman protocol, the amount of RNA is 10ng in each 15ul RT reaction).Because the concentrations of my total RNA isolated from Huh7-mock and Huh7-HCV replicon are 498ng/microliter and 520ng/microliter, I diluted total RNA to 10 ng/microliter by adding 1 microliter total RNA to 497 and 519 microliter RNase-free water,respectively.What's wrong with the dilution?

-yueh76-

txyuan on Tue Aug 30 03:10:59 2011 said:


According to Taqman protocol, the amount of RNA is 10ng in each 15ul RT reaction. If you use non-diluted RNA to run qPCR, Ct will become small. Especially for internal control, sometimes its Ct will be lower than 20. That means too much template is in each reaction. If Ct of your target is around 25, I don't think it is necessary to increase the amount of template. Even you increase RNA, it will not change the relative expression. Actually, I think the efficiency of PCR and the quality of cDNA are two major factors that affect the reproducibility.
By the way, why do you ensure the relative expression should be over 2-fold? Do you get this information from other paper or microarray? If it is from microarray, maybe it is not 100% reliable.

Thank you for your suggestion.
This information about relative expression between 2 to 3 fold is from paper and taqman qPCR result.

-yueh76-

yueh76 on Thu Sep 22 05:39:26 2011 said:


pcrman on Tue Aug 23 01:56:27 2011 said:


What is the difference between these two cell lines--one expresses the miRNA?

It should not matter much whether you dilute your RNA or not as long as you use the same amount of RNA for your RT reaction. However, the amount of RNA should be in a reasonable range. It seems that the amount of RNA you used is very low and the RNA was too diluted. Can you tell use the exact amount of RNA you used in the RT reaction?

I used 10ng total RNA for miRNA RT reaction (according to Taqman protocol, the amount of RNA is 10ng in each 15ul RT reaction).Because the concentrations of my total RNA isolated from Huh7-mock and Huh7-HCV replicon are 498ng/microliter and 520ng/microliter, I diluted total RNA to 10 ng/microliter by adding 1 microliter total RNA to 497 and 519 microliter RNase-free water,respectively.What's wrong with the dilution?

Is the dilution that is wrong. You are using a 1 ng/ul instead of 10. 1 ul of 498ng/microliter= 489 ng. 489ng/489 ul total volume= 1 ng/ul. My advice here is to check your counts and avoid using very small volumes for dilutions, errors increase greatly!
Fiz

-Fizban-

.................avoid using very small volumes for dilutions, errors increase greatly!
thanks Fizban, but how should I dilute my total RNA?
What is your advice?
Thank you very much.

-yueh76-

Fizban on Wed Nov 9 13:29:37 2011 said:


yueh76 on Thu Sep 22 05:39:26 2011 said:


pcrman on Tue Aug 23 01:56:27 2011 said:


What is the difference between these two cell lines--one expresses the miRNA?

It should not matter much whether you dilute your RNA or not as long as you use the same amount of RNA for your RT reaction. However, the amount of RNA should be in a reasonable range. It seems that the amount of RNA you used is very low and the RNA was too diluted. Can you tell use the exact amount of RNA you used in the RT reaction?

I used 10ng total RNA for miRNA RT reaction (according to Taqman protocol, the amount of RNA is 10ng in each 15ul RT reaction).Because the concentrations of my total RNA isolated from Huh7-mock and Huh7-HCV replicon are 498ng/microliter and 520ng/microliter, I diluted total RNA to 10 ng/microliter by adding 1 microliter total RNA to 497 and 519 microliter RNase-free water,respectively.What's wrong with the dilution?

Is the dilution that is wrong. You are using a 1 ng/ul instead of 10. 1 ul of 498ng/microliter= 489 ng. 489ng/489 ul total volume= 1 ng/ul. My advice here is to check your counts and avoid using very small volumes for dilutions, errors increase greatly!
Fiz

.................avoid using very small volumes for dilutions, errors increase greatly!
thanks Fizban, but how should I dilute my total RNA?
What is your advice?
Thank you very much.

-yueh76-

yueh76 on Thu Nov 10 05:32:12 2011 said:


Fizban on Wed Nov 9 13:29:37 2011 said:


yueh76 on Thu Sep 22 05:39:26 2011 said:


pcrman on Tue Aug 23 01:56:27 2011 said:


What is the difference between these two cell lines--one expresses the miRNA?

It should not matter much whether you dilute your RNA or not as long as you use the same amount of RNA for your RT reaction. However, the amount of RNA should be in a reasonable range. It seems that the amount of RNA you used is very low and the RNA was too diluted. Can you tell use the exact amount of RNA you used in the RT reaction?

I used 10ng total RNA for miRNA RT reaction (according to Taqman protocol, the amount of RNA is 10ng in each 15ul RT reaction).Because the concentrations of my total RNA isolated from Huh7-mock and Huh7-HCV replicon are 498ng/microliter and 520ng/microliter, I diluted total RNA to 10 ng/microliter by adding 1 microliter total RNA to 497 and 519 microliter RNase-free water,respectively.What's wrong with the dilution?

Is the dilution that is wrong. You are using a 1 ng/ul instead of 10. 1 ul of 498ng/microliter= 489 ng. 489ng/489 ul total volume= 1 ng/ul. My advice here is to check your counts and avoid using very small volumes for dilutions, errors increase greatly!
Fiz

.................avoid using very small volumes for dilutions, errors increase greatly!
thanks Fizban, but how should I dilute my total RNA?
What is your advice?
Thank you very much.

Use more RNA. instead of diluting 1 ul in 498 use 10 uls (which is actually what you should have done initially). there's a formula to calculate dilutions Vi (initial volume)*Ci (initial concentration)= Vf (final volume)*Cf (final concentration). In your case
Vi * 498 ng/ul = 498 (1 RNA + 497 water) * 10 ng/ul which is Vi = 498 * 10/ 498 = 10 ul Vi (the volume of your total RNA to be used in 498 ul (10 + 488).
Personal consideration, please do not take it the bad way: dilutions are part of the bases. It puzzles me how you are supposed to do real time PCR without knowing how to dilute RNA. It's ok and perfectly fine to ask here (this is what a forum is supposed to do), but isn't there anybody there taching you everything you need to work?
bye
Fiz

-Fizban-