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#1 phosphate girl

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Posted 10 June 2010 - 01:26 AM

Hello,

I am stuck and confused once again with cDNA synthesis and could do with some advice. I have extracted RNA from my samples at a range of concentrations. I know want to dilute them so that they are all at the same concentration. I currently have concentrations ranging from 250 ng/ul to 475 ng/ul. I'd like to get all samples to a concentration of 500 ng/ul so that I can use 2 ul of each in my cDNA synthesis (so 1 ug of RNA per CDNA reaction)

Here is how I was going to do it:

for a sample which is 250 ng/ul

take 5 ul of this so have 1250 ng RNA (5 x 250)

Use V1 x C1 = V2 x C2 to calulate how much water I will have to add to get a 500 ng/ul solution:

5 (ul) x 1250 = V2 X 500

5 x 1250/500 = 12.5

So I would add 12.5 ul of water to the 5 ul of RNA extract (at 250 ng/ul) to get a solution of 500 ng/ul

I can then add 2 ul of this solution to my cDNA synthesis (so 1 ug of RNA) to give me approx. 1 ug of cDNA

With my more highly concentrated RNA extract e.g 400 ng/ul I would just add less to the dilution to give me the 500 ng/ul solution:

400 ng/ul extract, take 3 ul (1200 ng/ul)

3 x 1200/500 = 7.2 (add 7.2 ul water to 3 ul of extract to get a 500 ng/ul solution)

Am I correct?? I just want to be sure before I start diluting down my RNA.

Many Thanks

#2 BryanC

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Posted 10 June 2010 - 05:30 AM

Hello,

I am stuck and confused once again with cDNA synthesis and could do with some advice. I have extracted RNA from my samples at a range of concentrations. I know want to dilute them so that they are all at the same concentration. I currently have concentrations ranging from 250 ng/ul to 475 ng/ul. I'd like to get all samples to a concentration of 500 ng/ul so that I can use 2 ul of each in my cDNA synthesis (so 1 ug of RNA per CDNA reaction)

Here is how I was going to do it:

for a sample which is 250 ng/ul

take 5 ul of this so have 1250 ng RNA (5 x 250)

Use V1 x C1 = V2 x C2 to calulate how much water I will have to add to get a 500 ng/ul solution:

5 (ul) x 1250 = V2 X 500

5 x 1250/500 = 12.5

So I would add 12.5 ul of water to the 5 ul of RNA extract (at 250 ng/ul) to get a solution of 500 ng/ul

I can then add 2 ul of this solution to my cDNA synthesis (so 1 ug of RNA) to give me approx. 1 ug of cDNA

With my more highly concentrated RNA extract e.g 400 ng/ul I would just add less to the dilution to give me the 500 ng/ul solution:

400 ng/ul extract, take 3 ul (1200 ng/ul)

3 x 1200/500 = 7.2 (add 7.2 ul water to 3 ul of extract to get a 500 ng/ul solution)

Am I correct?? I just want to be sure before I start diluting down my RNA.

Many Thanks



It doesn't work that way. You are using total RNA 1200ng and not concentration 250ng/ul in your equation, "C" has to be concentration. What you are doing would be:

C1V1 = C2V2
Think about it as 1=initial and 2=final

(250ng/ul)(5ul) = (Xng/ul) (17.5ul) its 17.5 because in your example you are adding 12.5 to 5

x = 71.42ng/ul so you are actually diluting your RNA, thus lowering the concentration and not raising it.


You don't need to change the concentration of all of your entire RNA samples before cDNA synthesis.

If you want to use 1ug or 1000ng RNA in each cDNA reaction, you normalize this in the actual tube in which you setup your reaction.

Sample 1: 250ng/ul

(1000ng)/(250ng/ul) = 4ul for 1000ng

In your cDNA kit, for a 20ul reaction a certain amount will be enzyme and buffer or other reagents. The rest is RNA and H20.

Example:

4ul Buffer
2ul Enzyme
4ul RNA (250ng/ul)
10ul H20
=
20ul Total Rxn Volume


You just go through the same calculation for each RNA sample and treat each rxn different as far as volume of RNA and volume of H20. So setup all of your Rxns with RNA and H20 first(will always be 14ul in above example), then make a master mix of the rest of your reagents and add the needed volume to each tube(6ul in the above example).

Edited by BryanC, 10 June 2010 - 09:10 AM.


#3 phosphate girl

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Posted 10 June 2010 - 07:53 AM

Hello,

Many Thanks for your help on this, I see where I was going wrong. Unfortunately, I have to do a DNase treatment before I start the cDNA synthesis which makes the whole thing more complicated:

for DNAse treatment it's a 10 ul reaction so:

4 ul of RNA (1000 ng)
1 ul DNase
1 ul buffer
4 ul water

This will give me 1000 ng of treated RNA in 10 ul so this will dilute the RNA again. Ideally I want 1000 ng of RNA in the cDNA reaction but I cannot add all 10 ul of the DNase treatment. 1000 ng in 4 ul into another 6 ul of reaction volume will give me 167 ng/ul ???


Ideally I want to add just 4-5 ul of the treated RNA max. Am guessing the only way I can do this is up the amount of RNA that goes into the DNase reaction? Have I completely lost the plot here?? :P If you could clarify this for me I would be very grateful.

#4 Trof

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Posted 10 June 2010 - 08:12 AM

I would definitelly measure RNA concentration again after DNAse treatment before you put it in the RT reaction. DNase treatment can degrade your RNA a bit.
We treat the whole RNA sample or do it on column while isolationg RNA prior to elution, then measure concentration and calculate how much to take for 1 ug. Store the rest DNase treated.

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#5 phosphate girl

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Posted 10 June 2010 - 08:45 AM

Hello,

Many Thanks for your post - I tried measuring the DNase treated RNA on a nanodrop and got very odd results. I blanked the nanodrop with a DNase reaction with with water in place of RNA (buffer, DNase, water). I just got loads of peaks instead of a nice single curve as I get with my pure extracts.....any suggestions???

#6 BryanC

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Posted 10 June 2010 - 09:04 AM

What protocols/kits are you using for RNA isolation and cDNA synthesis?

Edited by BryanC, 10 June 2010 - 09:04 AM.


#7 phosphate girl

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Posted 11 June 2010 - 01:05 AM

Hello Many thanks for you reply

I am using Promega RQ1 RNase-Free DNase - which I think is a firly standard protocol (add enzyme to your RNA with buffer and water, 10 ul reaction) incubate for 30 minutes at 37oC, add 1 ul of stop solution and incubate at 65oC for 10 minutes to inactivate the DNase. The protocol then says I can add all, or some of this straight in RT-PCR reaction.

For the RT-PCR I am using Roche Transcriptor High Fidelity cDNA Synthesis kit. I don't really want to add more than about 4 ul of the treated RNA (hence my concerns with dilution) to the RT-PCR because I will be carrying over Mg from the DNase buffer. Whilst I am reducing the quanity of RT-PCR buffer I add to the cDNA reaction to account for this, I don't want to adjust the quantities too much. I hope this makes sense??

It's turning out be rather complicted sadly :(

#8 phosphate girl

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Posted 11 June 2010 - 01:08 AM

Sorry forgot to mention the RNA extraction protocol - I used a modified protocol with the RNeasy mini kit to extract the RNA. I skipped the on column DNase step as the bulk of the samples were being sent for microarray (and apparently the DNase would interfere with this). Hence the need to do a seperate DNase treatment on these samples.

Cheers

#9 BryanC

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Posted 11 June 2010 - 05:38 AM

I can't really comment on DNase having an effect on microarry results, maybe someone else can clarify that.

I don't know if this is an option for you for future RNA isolations, obviously not in this case since you already have your RNA. In our lab, we use the Qiagen RNeasy Plus Mini Kit. It is supplied with a gDNA eliminator spin column and no DNase treatment is needed.

As far as [Mg2+] affecting downstream applications.

From your DNase kit's information sheet:

Notes:

1. Use 1 unit of RQ1 RNase-Free DNase per microgram of RNA. For smaller amounts of
RNA, use 1 unit of RQ1 RNase-Free DNase per reaction.

2. The RQ1 RNase-Free DNase digestion contains a final concentration of 10mM
MgSO4. When adding the DNase-treated RNA to an RT-PCR reaction, carryover of
magnesium must be considered. For example, the addition of 1μl of treated RNA to a
50μl RT-PCR reaction will raise the magnesium concentration by 0.2mM, and the
addition of 5μl of treated RNA will raise the magnesium concentration by 1mM. The
requirement for magnesium may be different in the RQ1 DNase digestion step and in
the amplification reaction.

• RQ1 DNase activity increases as the Mg2+ concentration increases up to
5–10mM. At a concentration of 1mM Mg2+, RQ1 DNase is expected to be at
least fourfold less active than at the optimal Mg2+ concentration.

• For some templates, the yield from the amplification reaction is highly
dependent on the Mg2+ concentration, and the optimal Mg2+ concentration
may be as low as 1mM.



If an increased Mg2+ concentration is not tolerable in the amplification reaction, the
following alternatives may be used.

• The RQ1 RNase-Free DNase 10X Reaction Buffer may be diluted 1:10 with
400mM Tris (pH 8.0), 10mM CaCl2 prior to DNase digestion. (Note that,
under these conditions, the RQ1 DNase will be approximately fourfold less
active than under standard reaction conditions.)

• An alternative DNase reaction buffer may be used (such as the RT or PCR
reaction buffer) if that buffer contains at least 1mM Mg2+.
• The RNA sample may be diluted in water prior to RT-PCR allowing dilution of the
MgSO4 to a concentration that is compatible with this application.

• The RNA may be purified with a standard phenol:chloroform extraction
followed by an ethanol precipitation.


Edited by BryanC, 11 June 2010 - 05:57 AM.


#10 phosphate girl

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Posted 14 June 2010 - 12:35 AM

Hello,

Many thanks for your reply, yeap I have reduced the amount of buffer used in my qPCR reaction to account for the carry-over as advised by the DNase protocol. Have decided to measure the RNA concentration after DNAse treatment and just add as much as I need to the qPCR reaction to give me 1 ug of RNA. I just think it will take some optimising to get the template concentration and Mg concentration right. Thanks again for your advice :)




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