cDNA synthesis with SYBR green (Biorad reagents) - KravitzLab/KreedLabWiki GitHub Wiki

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Calculate the amount of RNA you need for your cDNA synthesis

So this part is really dependant on your sample, how much RNA you will get from the extraction and the use for it. It's best to use a lot of RNA to be sure to detect low-expressed genes. Biorad recommend using up to 1000ng of RNA to make the cDNA, anything from 100ng to 1000ng will work fine, we will base the following calculations and explanation on using 500ng of RNA for the qPCR.

The reaction mix for cDNA synthesis will be 20 uL in total.

  • 1 uL of the reverse transcriptase Enzyme
  • 4 uL of the Reverse transcriptase mix
  • x volume of RNA
  • 20-(1+4+x) amount of RNAse free water.

Ex : You have around 56 ng/uL of RNA concentration in your sample. You will therefore need 500/56 = 8.92 uL of RNA to have a total quantity of 500ng of RNA And thus you will need 20-(8.92+1 (Enzyme) +4 (Mix)) = 6.08 uL of RNASe free water.

Step by step protocol

  • Put the amount of RNAse free water in your qPCR microcentrifuge tube (Ideally sterile, use gloves !)
  • Put the 4uL of Mix (KEEP THE MIX IN THE ICE BOX)
  • Put the 1uL of Enzyme (KEEP THE enzyme IN THE ICE BOX)
  • Add the x volume of RNA : For this step, keep the RNA ON ICE and gently do some up and downs with the pipette to mix it. Use Fileted tips and wear gloves. The RNA should be pure and stable but try to be as clean and thorough as possible.
  • Centrifuge the tubes for a few seconds to make sure all the drops are well into the well.
  • Set the tubes in the Thermocycler we have. For Biorad reagents we use the following program

5 minutes at 25°c 30 minutes at 42°c 5 minutes at 5°c

The program is present on the biorad thermocycler :

  • Put your tubes in
  • Close the Block A or B lid and tighten it
  • Click on "Run"
  • Go in "Admire"
  • Click on "BIORADFS"
  • Select the volume (20 uL)
  • Select the Block (A or B, where you put your tubes basically)
  • Select "Run"

The thermocycler will make start to run. The tubes can be taken out after 40 minutes. The PCR tubes can be kept at room temperature without any issues because cDNA is VERY stable even at room temperature. For caution, I recommend keeping them at -20°c for long term storage but you do not need to take any particular precaution when handling them.

Diluting the cDNA

So this step is optionnal. You do not need to obligatory dilute your cDNA, you can use the cDNA you have as is for the PCR. However, diluting the cDNA can be quite advantageous in most of context.

  • You get more materials to run in PCR, that can be great when you have a lot of genes to run
  • Diluting the cDNA can help to limit the amount of salts/phenol present during the qPCR, therefore optimizing the reaction.
  • It doesn't affect expression of very-highly expressed genes (See point below).

Effect of cDNA dilution on qPCR

In qPCR, the value for expression is the "Cq" value (Cycle quantification), it's basically says "How many cycles do I need to amplify my gene in order to detect it", the lower the value is, the more expressed the gene is. This value is logarithmically linked with the amount of cDNA you have in your preparation. Depending on how you dilute, it can affect the Cq value

Cq value (Diluted) = Cq value + Log 2 (Dilution factor).

If you dilute the cDNA 2 times

Cq value (Diluted) = Cq value + Log 2 (2)

Cq value (Diluted) = Cq value + 1

So if you were expecting a Cq value of 22 with a non diluted cDNA, it will be 23 with a dilution of 1/2

If you dilute the cDNA 5 times

Cq value (Diluted) = Cq value + 2.3

So if you were expecting a Cq value of 22 with a non diluted cDNA, it will be 24.3 with a dilution of 1/5

If you dilute 10 times

Cq value (Diluted) = Cq value + 3.3

So if you were expecting a Cq value of 22 with a non diluted cDNA, it will be 25.3 with a dilution of 1/10

For all qPCR, we are using a dilution of 1/5. Is that a big of a deal ? Depends on what you are looking for. If you are looking at well-expressed genes (Cq value between 20 to 30) it shouldn't pose any problem at all, especially if you dilute up to 1/5 because the values will be shifted to 22-32.

However, if you are looking at poorly expressed genes (Cq value above 30-32), it can be more problematic because it will move the Cq values of the genes to a 32-34 range which can be considered marginally expressed.

For that reason, its advised to always keep a stock of undiluted cDNA and a stock of diluted cDNA

Protocol to dilute the cDNA to 1/5

  • 1 : Take 16 uL of RNAse free water and put it in a new microcentrifuge tube
  • 2 : Take 4uL of your cDNA and put it in the previous tube
  • 3 : Gently do some ups and downs with the pipette to mix it
  • 4 : Centrifuge it for a few seconds.