cDNA synthesis

For the cDNA synthesis you need 0.5 µg RNA per sample. Using your obtained RNA concentration calculate the amount of µL that you need from your sample in order to obtain 0.5 µg RNA.

Have the calculation checked by the assistant!

• Mark one RNase-free PCR tube for eache sample + no RT controle.
• Note: these are special SMALLER tubes!
• Take the calculated volume from your RNA sample and pipette this amount in every RNase-free PCR tube (total RNA is then 0.5 µg per tube)
• Add 1 µl oligo dT
• The final volume in each RNase-free PCR tube should be 13.75µL calculate the amount of RNase-free milli Q that should be added to each RNase-free PCR tube
• Have the calculation checked by the assistant
• Add the calculated amount of milli Q in each PCR tube

Pipette a noRT-controle in the tube marked as noRT-controle:

•  Make an extra (duplo) for one of the DMSO conditions (1 cell line). Pipette the previously chosen V_RNA (for 0.5 µg), RNAse-free Milli Q and 1 µL oligo dT together.

NB: Where is this controle for? If you have read the protocol neatly in prepare mode, you will know this.

• Vortex and centrifuge the PCR samples briefly.
• Place the 9 samples in the PCR machine and start the RT program.
  • 5 minutes at 70 ° C
  • Then the samples are cooled to 4 ° C

 For the no RT-controle, add to your control tube:

• 4 µL 5x reaction buffer
• 2 µL 10 mM dNTP’s
• 0.25 µl RNAse-free Milli Q (instead of 0.25 µL reverse transcriptase!)

Make a “master mix” stock solution for the other samples.

• 4 µL 5x reactiebuffer
• 2 µL 10 mM dNTP’s
• 0.25µL RevertAid^tm M-MuLV reverse transcriptase

• Vortex the master mix stock solution for 3 sec.
• Add 6.25 µL master mix to each sample (not for the no RT control!).
• Resuspend all 9 samples (including the no RT contole).

 Put the 9 samples back into the PCR device and continue the RT program:

• 5 minutes at 37°C
• 60 minutes at 42°C
• 10 minutes at 70°C,
• Cool down to 4 °C

For samples with less than 0.5 µg of RNA added in the first step:

Calculate the amount of Milli Q to be added so that the final concentration of cDNA matches the other samples. Consider the reaction volume of the RT-PCR reaction and have your calculation checked by the assistants before adding the Milli Q.

Transfer the diluted sample to a 1,5 ml eppendorf tube, clearly labeled with:

• cDNA
• Group number
• Team number
• Sample (number)

The following calculation is aimed at samples up to 100 ng/µL.

Which of the following calculations is/are correct (more answers are possible)?

Assume the concentration of your sample is 1300 ng/µL and that the available volume in which the RNA is dissolved in Milli-Q is 20 µL (=desired/available total volume).

What volume of RNase-free Milli-Q needs to be added?

1. C1 * V1 = C2 * V2
   1300 ng/µL * ? = 100 ng/µL x 20 µL (RNA dissolved in Milli-Q)
   ? = 1,5 µL; add this volume to the RNA dissolved in Milli-Q
   Correct = no
2. C1 * V1 = C2 * V2 1300
   µL * 20 µL (RNA dissolved in Milli-Q) = 100 ng/µL * ?
   ? = 260 µL; add this volume to the RNA dissolved in Milli-Q.
   Correct = no
3. C1 * V1 = C2 * V2
   1300 ng/µL * 20 µL (RNA dissolved in Milli-Q) = 100 ng/µL * ?
   ? = 260 µL; add 260-20 = 240 µL to the RNA dissolved in Milli-Q.
   Correct = yes
4. Dilution factor = (1300 ng/µL) / (20 ng/µL)(RNA dissolved in Milli-Q) = 65
   1/65 * ? = 20 µL
   ? = 1300 µL; add 1300 – 100 = 1200 µL to the RNA dissolved in Milli-Q
   Correct = no
5. Dilution factor = (1300 ng/µL) / (100 ng/µL) = 13
   1/13 * ? = 20 µL (RNA dissolved in Milli-Q)
   ? = 260 µL; add 260-20 = 240 µL to the RNA dissolved in Milli-Q.
   Correct = yes

Feedback if correct: Correct! There are two ways in which this calculation can be done correctly. Using the C1 * V1 = C2 * V2 (C = concentration, V = volume, 1 and 2 referring to the concentrations and volumes that we have and we want with one unknown value), and by using a dilution factor in the calculation, where we divide the two concentrations and use 1/dilution factor * end volume formula

Feedback if a: Incorrect! The unknown value in this question is V2. The known concentration of your sample is 1300 ng/µL, the concentration you want is 100 ng/µL. We also know that the available volume in which the RNA is dissolved in Milli-Q is 20 µL (V1)

Feedback if b: Incorrect. In this calculation V2 = 260 µL. However, this is the end volume and not the volume we need to add. V2 should be the amount of RNA-free Milli-Q we need to add, which is calculated by the end volume – available volume in which the RNA is dissolved in Milli-Q.

Feedback if d: Incorrect. In this calculation the wrong concentration is used for the dilution step.

For cDNA synthesis we have diluted the samples to a RNA concentration of 100 ng/µL (see previous question). It is however possible that your sample already had a lower concentration than 100 ng/µL.

The next step is to calculate the volume of sample needed in order to have an amount of 0.5 µg RNA to insert into the qPCR machine. This amount is referred to as the V_RNA.

Which of the following calculations is/are correct (more answers are possible)? Keep in mind that a maximum of 12.75 µL RNA can be used for the cDNA synthesis.

1. Concentration = 45 ng/µL, total RNA needed = 0.5 µg. V_RNA = 0.5/0.045 µg/µL = 11.1 µL
   Correct = yes
2. Concentration = 100 ng/µL, total RNA needed = 0.5 µg. V_RNA = 0.5/1 µg/µL = 0.5 µL
   Correct = no
3. Concentration = 30 ng/µL, total RNA needed = 0.5 µg. V_RNA = 0.5/0.03 µg/µL = 16,7 µL
   Correct = no
4. Concentration = 100 ng/µL, total RNA needed = 0.5 µg. V_RNA = 0.5/0.1 µg/µL = 5 µL
   Correct = yes

Feedback if correct: Correct! By calculating and then diluting all of the samples to 100 ng/µL in the previous question we standardized all samples. For each sample with this concentration we will need to add a set amount of µL = VRNA in the PCR Eppendorf for the subsequent experimental steps. This means that this calculation depends on your previous lab work. As the total RNA needed for the qPCR experiment is 0.5 µg, the set amount is 50 µL by dividing the µg by µL to keep the right entity. However, sometimes the original RNA concentration is already below 100 ng/µL and then this set amount does not apply

Feedback if c: Incorrect. The total amount exceeds the maximum amount that can be used for cDNA synthesis

Feedback if b: Incorrect. There is a mistake in this calculation regarding a decimal point.

Waarom is het nuttig om een no-RT controle sample mee te nemen?

1. Dit sample wordt gebruikt om je andere samples te corrigeren voor vervuiling.
   • Juist! In de RNA isolatie kan er vervuiling opgetreden zijn van het RNA met DNA of andere eiwitten/celonderdelen. Wanneer je een sample meet waaraan geen reverse transcriptase is toegevoegd, meet je geen cDNA, maar DNA wat is meegekomen uit de RNA isolatie. Wanneer je je andere samples corrigeert met dit controle sample, corrigeer je de samples voor vervuiling.
2. Dit sample wordt gebruikt om na te gaan of de cDNA synthese goed gelukt is.
   • Incorrect. Wat bevat een no-RT controle sample? Kun je daarmee controleren of cDNA synthese goed gelukt is?
3. Dit sample wordt gebruikt om na te gaan of het proces in de PCR machine goed is verlopen.

o   Incorrect. Wat bevat een no-RT controle sample? Kun je daarmee controleren of het proces goed verlopen is?

Waarop is de keuze voor Oligo dT als primer gebaseerd? Er kunnen meerdere antwoorden goed zijn.

1. Deze primer is geschikt om cDNA te synthetiseren van uitsluitend de genen van interesse.
   Correct = no
2. Deze primer bindt specifiek aan de polyA staart van mRNA, waardoor cDNA gesynthetiseerd kan worden.
   Correct = yes
3. Deze primer brengt de synthese op gang, welke gekatalyseerd wordt door het enzym reverse transcriptase.
   Correct = yes

Feedback if correct: Juist! Oligo dT bindt aan de polyA staart van mRNA waardoor cDNA synthese plaats kan vinden, gekatalyseerd door het enzym reverse transcriptase.

Feedback if incorrect: Wil je hier de genen van interesse al selecteren?