RNA isolation, cDNA synthesis, and quantitative RT-PCR

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RNA extraction protocol with Qiazol/Trizol 

Reagents needed: 

-samples 

-Eppendorf tubes; 2ml round bottom and 1.5 ml (autoclaved) 

-chloroform 

-Qiazol lysis reagent 

-DEPC treated water (eliminates RNAse contamination) 

-75% Ethanol 

-isopropanol 

-ice & liquid nitrogen 

-forceps and scissors 

Protocol: 

1) Prepare all the solutions and tubes 

2) Take samples out of the freezer onto ice or liquid nitrogen, keep the samples on ice until homogenization 

3) Add Qiazol reagent (500ul per 50mg of tissue) into 2ml round-bottom eppendorf tubes (not conical) 

4) Add the samples to the tubes 

5) Homogenization 

-using blade and the UltraThurrax 

-or add one bead per tube and do the homogenization using the TissueLyser downstairs (for two minutes) 

6) After homogenization, incubate the samples at room temperature (RT) for 5min 

7) Add 100ul chloroform per 500ul Qiazol reagent, and shake the samples for 30s by inverting the tubes up-and-down 

8) Incubate at RT for 5min 

9) Centrifuge the samples 12 000 x g (rcf), 15 min, +4C (remember to precool the centrifuge) 

10) Carefully pipette the upper (clear) phase into a new 1.5 ml eppendorf tube. Avoid touching the white interphase with pipette. 

11) Add 250ul of isopropanol (per 500ul Qiazol used) and vortex 

12) Incubate at RT for 10 min 

13) Centrifuge 12 000 x g (rcf), 10 min, +4C 

14) Remove the supernatant and add 500 ul of 75% ethanol to the pellet. 

15) Vortex the sample and then centrifuge 7500 x g, 5min, +4C 

16) Remove the supernatant and let the pellet airdry about 10min RT 

17) Dissolve the pellet in RNAse free water (roughly 1 ul per mg tissue) 

18) Vortex gently and spin down the sample 

Measuring RNA concentration with Nanodrop 

1) Turn on the computer and Nanospec-software. Remove the black cover from the device. 

2) Select the 0.2 ‘valoaukko’ 

3) Pipette first 1ul of blank sample (RNAse free water) onto the black dot and measure 

4) Then name your own samples and measure them in duplicate 

5) Save the results in your own folder 

6) Store the samples at -70C until cDNA conversion 

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 RNA (Tissue) Extraction with Direct-zol™ RNA MiniPrep 

1. Add 500-700 uL of Qiazol (/TRI Reagent) and a metal bead in a 2ml round-bottom Eppendorf tube. Keep the tubes on ice and add tissue pieces (<25mg) into the tubes. (I used 600 ul) 
2. Homogenize with a Tissue Lyser for 3 min with 50 oscillation (50 is the max). 
3. Remove particulate debris: Centrifuge at 5 000 x g/rcf for 30 s at RT. 
4. Transfer the supernatant into a new RNase-free tube and let it equilibrate at RT. 
5. Add an equal volume of ethanol 600 ul (95-100%) to the supernatant and mix thoroughly by pipetting up & down. 
6. Transfer 500-700 ul of the mixture into a Zymo-Spin™ IIICG Column in a Collection Tube and centrifuge at 16,000 x g/rcf for 30 secs at RT. 
7. Discard the flow-through and transfer the remaining 500-700 ul of the mixture into the spin-column. 
8. Transfer the column into a new collection tube and discard the flow-through. 
9. DNase I treatment (in-column) 

(D1) Add 400 μl RNA Wash Buffer to the column and centrifuge at 16,000 x g/rcf for 30 secs at RT 

(D2) In an RNase-free tube, add 5 μl DNase I (6 U/μl)* & 75 μl DNA Digestion Buffer (per each sample) and mix. Calculate the total amount needed and add some extra for pipetting loss. 

Add the mix directly to the column matrix. 

(D3) Incubate at RT (20-30°C) for 15 minutes. 

1. Add 400 μl Direct-zol™ RNA PreWash to the column and centrifuge at 16,000 x g/rcf for 30 secs. at RT. Discard the flow-through and repeat this step. 
2. Add 700 μl RNA Wash Buffer to the column and centrifuge for 2 minutes at 16,000 x g/rcf to ensure complete removal of the wash buffer. 
3. Centrifuge again for 1 min at 16,000 x g/rcf to ensure that the column is completely dry. 
4. Transfer the column carefully into an RNase-free tube. 
5. To elute RNA, add 50 μl of DNase/RNase-Free Water directly to the column matrix, incubate at RT for 5 min and centrifuge at 16,000 x g/rcf for 30 secs.** 
6. Measure RNA concentration with NanoDrop and store the RNA samples at -20°C (short-term) or at -70°C 

*Store reconstituted DNAse I in small aliquots at -20°C 

***50 μl of DNase/RNase-Free Water for highly concentrated RNA 

N.B: Perform all steps at room temperature and centrifugation at 10,000-16,000 x g for 30 seconds, unless specified. 

Troubleshoot: Low RNA Yield/Concentration 

Tissue size – preferably less than 25mg 

Use 600uL of Qiazol and equal volume (600uL) of 100% Ethanol 

Remove particulate debris: Centrifuge at 5,000 x g/rcf for 20 sec. at room temp. 

Elute RNA with 25 μL of DNase/RNase-Free Water 

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PROTOCOL for cDNA reverse transcription with TAKARA Primescript RT kit 

Reagents needed: 

-small eppendorf tubes (0.2ml) 

-RNA-samples 

-cDNA reverse transcription kit / Primescript RT kit (stored at -20C at the DNA-free lab) 

-nuclease free water 

-filter tips 

-2 boxes of ice 

Preparations before starting: 

1) Based on your RNA-concentration values, calculate how much RNA-sample (in ul) you need to do a conversion for a total of 1 ug RNA (this is the maximum amount what can be converted) 

• Export your results from the BioSpec Nano computer into your USB-stick 
• Calculate first the average of your duplicate measurements 
• Depending on the total amount of RNA you want to convert into cDNA (typically 1 ug or less), divide the total RNA value with the RNA concentration of your sample 
• For example: if you are aiming to convert 1 ug and the RNA conc. of your sample is 500 ng/ul -> V (ul)=1000ng/500ng/ul ->2,0ul 
• Do this calculation for all of your samples 
• The maximum volume of a sample what you can pipette into a reaction is 5ul. 
• The volume of your sample is finally adjusted to 5ul, so you need to also calculate how much water you need to add to each of your sample to have a total volume of 5ul in all the samples 
• For the abovementioned example: V (H20) = 5ul-2,0ul=3,0ul 
• Do a pipetting chart for your samples 

2) Label your Eppendorf tubes for the RT reaction 

3) Put your RNA samples on ice for thawing in the bigger PCR lab 

Step 1: Preparation of the RT master mix in the DNA-free lab (smaller lab) 

1) Go the lab well in advance, to put the components of the cDNA kit on ice for thawing 

2) Pipette the RT master mix by combining the 4 components. Calculate the total amount and put about 1-2 sample extra for pipetting loss 

3) Vortex gently the master mix and spin it down, and keep on ice thereafter 

Step 2: Preparation of RT reactions in the bigger PCR lab 

1) Pipette the RNA samples into the small PCR tubes and adjust the volume to 5ul 

2) Pipette the RT master mix into the tubes (5 ul per tube) 

-5ul sample+water and 5ul RT mix -> total reaction volume 10ul 

3) Close the tubes and centrifuge quickly to get the solution to the bottom 

4) Put the reaction tubes on ice again 

Step 3: Reverse transcription 

1) Program the thermal cycler according to the table: 

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 Protocol for quantitative RT-PCR 

Reagents: 

DNA-free lab 

• Ice box 
• 1,5 ml eppendorf tubes (autoclaved) 
• filter tips 
• SYBR reagent 
• FW and REV primers 
• Nuclease free water 

PCR-lab 

• Ice box 
• Filter tips 
• 96-well plate(s) 
• adhesive film 
• cDNA samples 

1. Preparation of the master mix in the DNA-free lab 

• Prepare own master mix for each primer pair separately according to the pipetting chart 
• Vortex and spin down the master mix, thereafter keep on ice 

2. Preparing the plate 

• Put the 96-well plate on ice 
• Mix your sample by vortexing and then pipette in the well (3ul per well). Pipette the samples in duplicate. 
• Pipette the master mix then into each well (17ul per well) 
• Seal the plate with adhesive film 
• Centrifuge the plate quickly with the plate centrifuge (let the RPM value increase to 1000 and then stop the centriifugation). 
• Put the plate again on ice thereafter 

3. Setting up the qPCR run 

1. Switch on the qPCR machine before the run (5-10 min before). 
2. Create a new document 
3. Assay → New ddCt plate. 
4. Select SYBR green as detector → Add 
5. Select the wells and active above SYBR. 
6. Name your samples/wells 

2 

1. Instrument-sheet 
2. Remove Step 1 
3. Enzyme activation 95 °C, 3:00 min 
4. Denature 95 °C, 0:03 min 
5. Anneal 60 °C, 0:27 min 
6. Add dissociation stage 
7. Set reaction volume 20 μl 
8. Save the file 
9. Set in your plate 
10. Start 
11. After the run, remove the plate from the machine and discard it 

Setting for the new Sensifast SYBR reagent: 

Enzyme activation: 95 ℃ 2min 

Denature : 95℃ 5sec 

Anneal : 60℃ 30 sec