RNA expression of gene encoding for Cx43 protein from qPCR

Theory

tessa There are different types of Polymerase Chain Reactions (PCR) one of which is quantitative PCR (qPCR), used to quantify the amount of RNA molecules within a sample, relative to the samples analyzed, whereas PCR is used to analyze, or amplify, the presence of DNA. [54] By analyzing whether certain gene strands are present and in which ratio, information is gathered on the ability of a sample to express certain proteins. It should be taken into account that qPCR alone does not give information on the expression of the proteins, only the available opportunity for a sample, or cell, to facilitate this expression.

RNA has to be transcribed to cDNA, a process which is called reverse transcriptase, after lysating a tissue sample to gather RNA and isolating said RNA, as RNA is easily degradable. After the RNA is isolated, cDNA has to be formed. To allow for as little as possible inter-sample technical differences, the amount of RNA set to be transcribed into cDNA should be equal among all samples. For this, the amount of RNA in ng/mL is measured with a nanodrop. First the RNA is denatured, after which a primer binds to the RNA strand, reverse transcriptase binds deoxyribose nucleoside triohosphates (dNTPs), the building blocks of DNA a process called annealing, creating cDNA. Each step of this process is heat dependent and is therefore done by a thermocycles. As cDNA does not disintegrate as rapidly as RNA, the cDNA can be stored at -20 ◦C for months.

With the cDNA, qPCR can be performed. First, the cDNA is denatured to allow for annealing. Afterwards, the strands separate, having created two copies of an identical cDNA strand. This process is repeated 20-40 times for the purpose of creating enough cDNA for detection. In qPCR, fluorescent markers bind to specific gene strands in the cDNA. The fluorescence is measured and can be compared to measured fluorescence in other samples. qPCR is therefore highly qualified to compare between samples. [55]

Methods RNA isolation

This protocol has been adapted from the RNeasy Mini kit from Qiagen.

• Add 350 µL RLT buffer to each sample and slowly pipet up and down to lysate the cells. Gather the RLT buffer with cells.

• Add 350 µL pure ethanol to the RLT buffer and mix well by pipetting.

• Transfer the 700 µL to an RNeasy Mini spin column placed in a 2 mL collection tube.

• Centrifuge this with the eppendorf Centrifuge 5810 R for 15 sec at >8000 g and discard the flow through.

• Add 700 µL Buffer RW1 to the spin column.

• Centrifuge this for 15 sec at >8000 g and discard the flow through.

• Add 500 µL Buffer RPE (with ethanol) to the spin column.

• Centrifuge this for 15 sec at >8000 g and discard the flow through.

• Add 500 µL BufferRPE (with ethanol) to the spin column.

• Centrifuge this for 2 min at >8000 g and discard the flow through.

• To dry the membrane centrifuge the spin column again for 1 min at >8000 g and discard the flow through.

• Place the RNeasy spin column in a new 1.5 mL collection tube, the 2 mL collection tube can be discarded.

• Add 30 µL RNase-free water to the spin column.

• Centrifuge this for 1 min at >8000 g to elute the RNA.

• When the expected RNA yield is low, the previous 2 steps can be repeated with the eluate obtained.

cDNA formation

CHAPTER 4. EXPERIMENTAL METHODS

– Clean the nanodrop.

– Measure a blank sample.

– Measure each sample twice, be sure to wipe the nanodrop between samples.

– Calculate the average.

• Compensate to make sure each sample has the same amount of RNA by mixing variable amount of RNA template and nuclease-free water till 15 µL in a small tube.

• Add 4µL 5x iScript Reaction Mix and 1µL iScript Reverse Transcriptase.

• Incubate the tubes in a MJ MiniTM Personal Thermal Cycler with the following protocol:

– 5 minutes at 25 ◦C.

– 20 minutes at 46◦C.

– 1 minute at 95 ◦C.

– Store at 4 ◦C

This protocol is obtained from the iScript cDNA Synthesis Kit.

Performing the qPCR This protocol has been adapted from a protocol written, but unpublished, by Karlijn Slutter, based on a version of Verena Schwach, a version of which can be found on Benchling.

• First, thaw the primers on ice.

• Dilute cDNA with RNase free water in a 1:16 concentration (285 µL water + 19 µL cDNA).

• Dilute each primers to be used with RNase free water in a 1:10 concentration (90 µL water + 10

µL primer). These dilutions can be stored at -20 ◦C and reused.

• Prepare the Mastermix, take into account that each sample will be measured in triplo and that each primer will have a negative control (also in triplo), according to the following amounts:

– For each well add 5 µL SensiMix SYBR & Fluorescein Mix.

– For each well add 0.5 µL Forward Primer.

– For each well add 0.5 µL Reverse Primer.

• Take a 384 qPCR plate, remember not to talk in front of the plate to inhibit the chance of contam- ination.

• Add 6µL mastermix to each well.

• Add 4µL cDNA to each well or RNase free water to each negative control

• Cover the plate with a foil and make sure it sticks well to the plate with a Film Sealing Roller. Make sure not to touch the foil.

• Spin the plate for 30-60 seconds at 3000 g with the eppendorf Centrifuge 5424 R until air bubbles are gone.

• Place the plate in the CFX384 TouchTM Real-Time PCR Detection System and enter the following protocol:

1. 10 minutes at 95◦C. 2. 15 seconds at 95 ◦C. 3. 15 seconds at 60 ◦C. 4. 15 seconds at 72 ◦C. 5. Repeat step 2 for 39 times.

6. Add a melt curve from 65 degrees to 95 ◦C, with steps of 0.5 degrees. 7. End at 4 ◦C ’forever’, to keep the plate cool till further use.

• The plate can be used for gel electrophoresis, which was not performed in this master thesis, instead the plate was thrown.

• The qPCR should be repeated 3 times to compensate for technical errors, such as pipetting errors.

CHAPTER 4. EXPERIMENTAL METHODS