RNA

 

Beata Scholtz

 

University of Debrecen, Medical and Health Science Center, Department of Biochemistry and Molecular Biology, Genomics Center, Hungary, Tel.: +36 52 411717 ext.55001, Fax: +36 52 314989

[email protected]

 

 

Practical 1 - Isolation of RNA for the microarray experiments

 

Introduction:

You will isolate total cellular RNA from cell cultures for the microarray experiments. Since this is a time-course experiment, you will receive the lysed samples from the various time points, not the live cells, and will have to proceed from there with the protocol. It is very important that you wear gloves all the time, and change them frequently to minimize the risk of contaminating you samples with exogenous RNase.

 

 

Practical 1A: Isolation of total cellular RNA with the Qiagen RNeasy kit

 

At the end of this chapter you will find the details of the first part of the RNA isolation protocol, that produced the stabilized cell lysates for your experiment (“RNA isolation – Additional information”). Keep the cell lysates on ice until you are ready to start the procedure.

 

Checklist:

Cell lysates, in RLT buffer (on ice)

RNeasy mini columns

Sample collection tubes: 2 x 2 ml, capless, 1 x 1.5 ml, with cap

70% ethanol

RW1 buffer

RPE buffer

nuclease free water

 

1. Mark the RNeasy mini columns and the collection tubes (three tubes per sample) with the sample code numbers, and place the RNeasy columns in the 2 ml collection tubes.

 

2. Add 1 volume of 70% ethanol to the homogenized lysates and mix well by pipetting. Do not centrifuge. Visible precipitates may form after the addition of ethanol when preparing RNA from certain cell lines, but this will not affect the RNeasy procedure.

Volume of your lysates: 300 ml

 

3. Apply up to 700 µl of the sample, including any precipitate that may have formed, to the RNeasy mini column. Close the column caps and centrifuge the samples for 15 sec at

8000 g, room temperature, in the tabletop centrifuges.

 

4. Discard the flow-through. Place the columns back in the same collection tubes.

 

Note: this is the step when the RNA in your sample binds to the silica matrix of the minicolumns. However, the matrix still retains some DNA and proteins at this stage, which will be removed in the following washing steps with the RW1 and RPE wash buffers.

 

5. Add 700 µl RW1 buffer to the RNeasy mini column. Close the column caps and centrifuge the samples for 15 s at 8000 g, room temperature, in the tabletop centrifuges. Discard the flow-through and the collection tubes – transfer the columns into fresh collection tubes.

 

6. Add 500 µl RPE buffer to the RNeasy mini column. Close the column caps and centrifuge the samples for 15 s at 8000 g, room temperature, in the tabletop centrifuges.

 

7. Discard the flowthrough. Place the columns back in the same collection tubes.

 

8. Repeat the washing step: again, add 500 µl RPE buffer to the RNeasy mini column. Close the column caps and centrifuge the samples for 2 min at 8000 g, room temperature, in the tabletop centrifuges. Discard the flow-through and the collection tubes – transfer the columns into fresh collection tubes.

 

Note: The RPE buffer is supplied as a concentrate, and is diluted to working solution with ethanol. Since residual ethanol from the RPE buffer on the column will end up in your eluate, it may interfere with many downstream applications. Therefore, before sample elution, it is critical to remove the RPE wash buffer from the column as thoroughly as possible. Centrifuging for 2 min in step 8 serves that purpose – in addition, it is advised to perform a third centrifugation step – step 9.

 

9. Centrifuge the columns in a microcentrifuge at full speed for 1 min, room temperature.

 

10. Elution: Place the RNeasy columns in new 1.5 ml Eppendorf tubes. Pipet 50 µl nuclease free water directly onto the RNeasy silica-gel membrane, but make sure that the pipette tip does not touch the membrane. Close the column caps and incubate the sample for 5 min at RT, then centrifuge for 1 min at 8000 g, room temperature to elute.

 

11. Remove the columns from the Eppendorf tubes, close the tubes and place them on ice. This is your total RNA preparation.

 

Practical 1B: RNA quantitation with the NanoDrop spectrophotometer

 

 

Introduction:

The various approaches used for RNA quantitation are based on either the specific light absorbance of the ribonucleic acid itself, at certain wavelenghts (detected by spectrophotometer), or on detecting the fluorescence of the RNA-bound form of specific dyes (RiboGreen reagent, or Agilent 2100 Bioanalyzer). An easy way to quantify even dilute RNA samples is to use the NanoDrop spectrophotometer.

The NanoDrop® ND-1000 is a full spectrum (220-750 nm) spectrophotometer. Nucleic acids have the highest absorbance at 260 nm – contaminating proteins or phenol have higher absorbance at 280 nm. Measuring the absorbance of your samples at 260 nm allows calculation of their concentration, whereas measuring the absorbance of your samples at 280 nm and 230 nm provides information about the purity of the samples.

 

The RNA is considered good quality if the R=A260/A280 ratio is close to 2.0. If lower than that, it usually means protein or phenol contamination. You can also use the 260/230 ratio to help determine the amount of organic contamination in your RNA. It is a much more variable number than the 260/280 ratio, but generally, ratios above 1 indicate good purity.

 

 

1. You will need 2 µl of the RNA sample for quantitation. The sample is measured directly, without dilution, since there is no cuvette for the NanoDrop. You will also need RNase-free water as the blank sample.

 

2. Blanking cycle: Load a blank sample (2 µl water) onto the lower measurement pedestal and lower the sampling arm into the “down” position.

 

3. Click on the “Blank” (F3) button.

 

4. Wipe the blanking buffer from both pedestals with a Kimwipe.

 

5. Analyze an aliquot of the blanking solution as though it were a sample - load a blank sample (2 µl water) onto the lower measurement pedestal and lower the sampling arm into the “down” position. Click on the “Measure” button (F1). The result should be a spectrum with a relatively flat baseline. Wipe the blank from both measurement pedestal surfaces and repeat the process until the spectrum is flat.

 

6. Analyze your sample: load 2 µl of the RNA sample onto the lower measurement pedestal and lower the sampling arm into the “down” position. Click on the “Measure” button (F1). Print the results.

 

7. Wipe the sample from the pedestals with a Kimwipe, then clean the pedestals by loading 2 µl water on them, and wiping again with a Kimwipe.

Practical 1C - RNA quality control with Agilent:

 

 

Introduction:

Part of the RNA quality control is determining the 260:280 absorbance ratio, which can warn about the presence of various contaminants. Since microarray experiments are especially sensitive to differences in RNA integrity, it is essential to use either gel electrophoresis, or, better, the Agilent 2100 Bioanalyzer to characterize RNA quality. The Bioanalyzer is used with the RNA 6000 Nano LabChip kit – samples are loaded onto the LabChip, which contains a special gel matrix with an RNA-intercalating dye in a miniature capillary system. When the capillary electrophoresis is performed, the dye binds to the double-stranded regions of RNA molecules, and as the molecules are separated by size and migrate through the capillary matrix, fluorescence of the samples is recorded continuously by the instrument.

 

 

 

Running the RNA 6000 Nano LabChip

 

 

1. Preparing the gel:

·      Put 550 ml of RNA Nano Gel Matrix into a spin filter.

·      Centrifuge at 1500g for 10 min.

·      Aliquot 65 ml of filtered gel into RNase-free tubes.

2.     Preparing gel-dye mix:

·      Vortex RNA 6000 Nano dye concentrate vigorously.

·      Spin down

·      Add 1 ml of dye into a 65 ml aliquot of filtered gel

·      Note that dye is photosensitive. Do not expose it to light for too long.

·      Vortex solution well.

·      Spin down at 13000 g for 10 min at room temperature.

3.     Loading gel-dye mix:

·      Put a new RNA Nano chip into the Chip Priming Station.

·      Pipette 9 ml of gel-dye mix into the third G marked well.

·      Close Chip Priming Station.

·      Press plunger until it the clip.

·      Wait exactly 30 sec - release the clip - wait an additional 30 sec.

·      Pipette 9 ml gel-dye mix into the other G marked wells.

4.     Loading RNA 6000 Nano Marker:

·      Pipette 5 ml of RNA 6000 Nano Marker in the marked wells (1 Ladder and 12 Samples).

5.     Loading Ladder and Samples:

·      Pipette 1 ml of RNA 6000 ladder into the marked well.

·      Pipette 1 ml of sample into each sample well.

·      Put the chip into the vortex with the adapter and vortex for exactly one minute at the indicated speed (2400 rpm).

·      Run the chip in the Agilent 2100 Bioanalyzer within five minutes.

6. Print and save results in PDF format (gel-like image, electrophoretogram, RIN calculation) for further analysis and discussion.

 

 

 

 

 

 
 
 

 

 

 



Practical 1D: DNase I treatment of total RNA

 

 

Introduction:

A portion of the RNA prepared for the microarray experiment will be DNase-treated. Total RNA samples are usually treated with DNase I prior to QPCR, to remove co-purified genomic DNA contamination. In many cases the QPCR assays are designed over exon-intron boundaries, and would not detect the gene in the genomic DNA; however, intronless pseudogenes (or intronless genes, or assays within a single exon) may still give a background signal.

 

Checklist:

RNase-free DNase (from the instructor)

10X reaction buffer with MgCl2

0.5 M EDTA

nuclease-free water

 

1. Combine the following components in a fresh 1.5 ml Eppendorf tube, on ice:

 

2. Incubate the samples at 37 °C for 20 min

 

3. Inactivate the enzyme:

 

RNA isolation – Additional information

 

 

A. From cells to stabilized lysates with the Qiagen RNeasy kit

 

The entire manual (“RNeasy mini handbook”) can be downloaded from the Qiagen homepage: http://www.qiagen.com

It is srongly advised that you read through the manual carefully before using the kit on your own – however, the most important information for the processing of the cells is summarized below.

 

There are three critical issues to keep in mind when processing the cells:

 

1) Keep the recommended cell number : lysis buffer (RLT buffer) ratio.

 

For suspension cultures, use 350 µl RLT lysis buffer for < 5 x 106 cells, and 600 µl RLT lysis buffer for 5 x 106 – 1 x 107 cells. Of course, you can make more lysate by proportionally increasing the volume of lysis buffer, but do not load lysates prepared from more than 1 x 107 cells for one RNeasy mini column. Also, keep in mind that the RNA binding capacity of the mini columns is 100 µg RNA. Larger kit formats are also available from Qiagen. If you use inappropriately low volumes of the RLT buffer, you risk incomplete lysis, as well as incomplete inhibition of endogenous RNases, which will adversely affect the quality of your samples.

 

2) Minimize cell handling before lysis.

 

Cellular stress and cell death may cause RNA degradation even before the stabilizing RLT lysis buffer is added to the cells. Therefore, washing the cells before lysis either in a suspension culture or in a monolayer culture is not recommended. Centrifuge the suspension culture and remove the cell culture medium thoroughly before adding the RLT lysis buffer to the cell pellet. For monolayer cultures it is better to lyse the cells directly in the culture dish, after removing the cell culture medium – use larger volumes of the RLT buffer to cover the surface of the dish.

 

3) Lyse the cells QUICKLY and thoroughly.

 

For cells grown in suspension, loosen the cell pellet thoroughly by flicking the tube, add the RLT buffer, vortex to mix, then homogenize the cells by passing the lysate through a 20-gauge needle fitted to an RNase-free syringe 5-10 times. RLT lysates can be stored at -70°C for several months.

 

 

 

Tips and Tricks: RNA isolation and quantification

 

 

 

 

 

 

 

 

 

c=(A x e)/b

 

Where c= concentration of the sample, in ng/µl.

A = is the absorbance of the sample, as measured

e= is the wavelenght-dependent extinction coefficient in ng-cm/µl

b= is the path length in cm.

 

The generally accepted extinction coefficient for RNA is 40 ng-cm/ml at 260 nm.

The measurement concentration range of the instrument is 5 ng/µl – 3000 ng/µl.

 

It should be noted that acidic solutions will under-represent the 260:280 ratio by 0.2-0.3, while basic solutions will over-represent 260:280 ratio by 0.2-0.3. Since pure water, such as distilled water, MilliQ water or RNase-Dnase free water has an acidic pH, more accurate measurements of the 260:280 ratio can be made if the nucleic acids are dissolved in buffered, pH=close-to-neutral solvents. In addition, the absorbance of nucleic acids plateaues around 260 nm, but shows a deep slope around 280 nm. Therefore, depending on the accuracy of nm settings for different spectrophotometers, one can experience consistently higher or lower 260:280 ratios when switching to a different instrument. To assess the true quality of your samples, it is better to record the full UV spectra, or at least take the 260:230 ratio as well. Some samples may appeare clean when just taking the A260 and 260/280 ratios. It only becomes clear that they have very different purities when a full UV spectrum is taken.

 

 

Tips and Tricks: Agilent Analysis

 

·      Filtered gel can be used within four weeks.

 

·      Always use fresh gel-dye mix.

 

·      Always check prime station position before using it.

 

·      Ladder must be denatured before first using it.

 

·      Check chip after loading gel into capillaries for bubbles.

 

·      If less than 12 samples are analyzed, fill empty wells with Marker and nuclease free water similarly as if they were in use.

 

·      Before and after using the Bioanalyzer wash electrodes with 300 ml of RNA ZAP and nuclease free water using the washing chips.

 

·      Place Agilent 2100 Bioanalyzer to a quiet bench. It is sensitive to any mechanical stress.

 

·      Catalogue number for Agilent RNA 6000 Kit: 5067-1511

 

 

Tips and Tricks: DNase treatment

 

 

·      Ambion provides an excellent summary of DNases and DNase I treatment of RNA at: http://www.ambion.com/techlib/tips/dnase1demystified.html

 

·      An important point to consider is how to inactivate the enzyme, which would otherwise interfere with downstream RT and PCR applications. Heat inactivation is convenient, but RNA undergoes spontaneous degradation at high temperatures if divalent cations are present. That’s why it is recommended to add EDTA to the samples before heat inactivation. If the samples are not purified further with precipitation or column-based purification, the final concentration of EDTA should be balanced to provide protection from degradation, but not to inhibit downstream enzymes also requiring divalent cations for their activities. This is usually achieved by treating more RNA in one reaction, and diluting the DNase-treated RNA appropriately for RT.

 

Reagents/solutions

 

 

Qiagen RNeasy mini kit

Supplier: Qiagen

Cat. No.: 74104

 

 

Trizol

Supplier: Invitrogen

Cat. No.:15596-018

 

Nuclease-free water

Supplier: Promega

Cat. No.: P1195

 

RNase-free DNase I

Supplier: Ambion

Cat. No.: 2222 (2000 U, 2U/ml)

Practical 2 - Preparation of RNA for the miRNA QPCR experiments

 

 

Introduction:

You will isolate total cellular RNA from cell cultures for the miRNA QPCR experiments. Since this is a time-course experiment, you will receive the lysed samples from the various time points, not the live cells, and will have to proceed from there with the protocol. This will be a Trizol-based purification – the Qiagen RNeasy columns cannot be used, since they let through the small-sized RNA fraction. In the „Tips and Tricks “ section you will find more detailed information about the RNA isolation protocol (#A). It is very important that you wear gloves all the time, and change them frequently to minimize the risk of contaminating you samples with exogenous RNase.

 

 

Practical 2A: Isolation of total cellular RNA with Trizol

 

 

At the end of this chapter you will find the details of the first part of the RNA isolation protocol, that produced the stabilized cell lysates for your experiment (“RNA isolation with Trizol – Additional information”). Keep the cell lysates on ice until you are ready to start the procedure.

 

Checklist

Cell lysates, in Trizol (on ice)

Chloroform

Isopropyl alcohol

75% ethanol

Nuclease-free water

 

 

1. Phase separation:

 

Following centrifugation, the mixture separates into lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase, while genomic DNA is in the lower phase, and precipitated proteins collect at the interface, as a whitish ring or layer.

 

2. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube. It may be better to transfer multiple small volumes, rather than trying to aspirate the entire upper phase and risking contamination with the interphase/lower phase. If this happens, however, you can repeat the centrifuging step to separate the phases again.

 

3. RNA precipitation:

 

4. RNA wash :

 

5. Redissolving RNA:

·      Air-dry the RNA pellet for 5-10 minutes under the flow of a chemical or sterile hood. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. The RNA pellet is dry enough if there is no visible liquid above the pellet, the pellet is sticking completely to the tube wall, and the pellet surface is still shiny, not dull white.

 

 

 

 

 

 

 

 

RNA isolation with Trizol – Additional information

 

 

A. From cells to stabilized lysates with Trizol reagent

 

The entire protocol can be downloaded from the Invitrogen homepage: http://www.invitrogen.com

 

Just like with the Qiagen RNeasy kit, there are three critical issues to keep in mind when processing the cells:

 

1) Keep the recommended cell number : Trizol reagent ratio.

 

If you use inappropriately low volumes of Trizol, you risk incomplete lysis, as well as incomplete inhibition of endogenous RNases, which will adversely affect the quality of your samples. It is better to use excess Trizol, especially if you experience problems with RNA integrity.

 

2) Minimize cell handling before lysis.

 

Cellular stress and cell death may cause RNA degradation even before the cells are lysed in Trizol. Therefore, wash the cells before lysis only once with ice-old PBS, or not at all. Centrifuge the suspension culture and remove the cell culture medium or PBS thoroughly before adding Trizol to the cell pellet. For monolayer cultures it is better to lyse the cells directly in the culture dish, after removing the cell culture medium or PBS – use larger volumes of Trizol to cover the surface of the dish. Tilt the culture dish back and forth a few times to aid complete cell lysis.

 

3) Lyse the cells QUICKLY and thoroughly.

 

For cells grown in suspension, loosen the cell pellet thoroughly by flicking the tube – make sure that there are no cell clumps left. Add Trizol, vortex to mix, then, if working with large enough volumes, homogenize the cells by passing the lysate through a 20-gauge needle fitted to an RNase-free syringe 5-10 times. With smaller volumes, homogenize by pipetting the sample up-and-down several times with a Gilson pipette.

 

4) Incubate the homogenized sample for 5 minutes at room temperature to permit the

complete dissociation of nucleoprotein complexes. Centrifuge to remove cell debris. Trizol lysates can be stored at -70°C for several months.

 

 

Tips and Tricks

 

 

 

 

 

 

 

 

Practical 3 - RT-IVT Labeling the RNA for the microarray experiment

 

 

Introduction:

We will use the Applied Biosystems microarray system to perform the microarray experiments. The system has three components: the Chemiluminescent RT-IVT labeling kit, the microarrays, and the Chemiluminescent Detection kit. The purpose of the Chemiluminescent RT-IVT labeling kit is to convert RNA into digoxigenin (DIG) labeled cRNA for hybridization. The DIG-labeled cRNA will bind to the oligonucleotide probes on the array surface in a sequence-specific manner, via complementary base-pairing. The DIG-residues in the cRNA are recognized by the DIG-specific antibody in the Chemiluminescent Detection kit. The DIG-specific antibody is coupled with alkaline phosphatase, and when its substrate added to the array surface, the chemiluminescence-producing reaction is initiated in the spots of the array where the antibody binds.

 

The Chemiluminescent RT-IVT labeling is a two-day procedure – on the first day you will complete Phase 1. This includes the following steps:

 

1. Reverse transcription of the RNA to cDNA, with a T7-oligo(dT) primer

Product: single-stranded cDNA

2. Second strand synthesis and purification of double-stranded cDNA

Product: double-stranded cDNA and degraded RNA

3. In vitro transcription (IVT) and DIG-labeling

Product: DIG-labeled cRNA, linearly amplified from cDNA. Since the IVT process requires a 9-hour incubation, the product will be ready by tomorrow.

 

Phase 2 (next day):

 

1. End of IVT labeling: Purification of cRNA

2. Assess the quality and quantity of the cRNA product

 

 

 

Practical 3A: Labeling the RNA with the AB Chemiluminescent RT-IVT labeling kit

 

 

The entire manual (Applied Biosystems NanoAmp RT-IVT Labeling Kit Protocol) can be downloaded from the Applied Biosystems homepage: http://www.appliedbiosystems.com

It is strongly advised that you read through the manual carefully before using the kit on your own – the protocol below is a summary.

 

 

A) Reverse transcription: 1st strand synthesis

 

Checklist:

RNA sample – 2 µg of total RNA dissolved in RNase-free water

T7-Oligo(dT) primer

Control RNA: three bacterial mRNA transcripts

Nuclease-free water

10X 1st strand buffer mix

dNTP Mix

RT enzyme mix

RNase inhibitor

 

1. Allow the frozen reagents to thaw on ice, vortex them, centrifuge the tubes briefly and place them back on ice. Keep the RNA solution on ice at all times. Keep the RT enzyme mix at –20 °C until the last minute, and do not vortex it.

 

2. Pipette the following components into a 0.2 ml Eppendorf tube, marked with the code number of your RNA sample:

 

·      1 µl T7-Oligo(dT) primer

·      2 µl Control RNA (diluted 1:5000 by the course staff)

·      maximum 9 µl of the RNA sample (2 mg)

 

Total volume: 12 µl

 

3. Heat and cool the RNA and primer mixture in a thermal cycler:

·      Program: 5 min at 70 °C (melting)

Indefinite hold at 4 °C (primer annealing)

·      Set the reaction volume to 12 µl

·      Load the tubes in the cycler and start the run

·      After the run is complete, place the tubes on ice

 

4. Check the 10X 1st strand buffer for precipitates (undissolved DTT). If precipitates are present, warm the buffer at 37 °C for 5 minutes, then vortex it briefly before using.

 

5. Add the following components to the reaction tube on ice and mix thoroughly by pipetting:

·      2 µl 10X 1st strand buffer

·      4 ml dNTP mix

·      1 ml RT enzyme

·      1 ml RNase inhibitor

 

6. Perform reverse transcription in the thermal cycler:

·      Program:

o      10 min at 25 °C (initiation)

o      2 hours at 42 °C (extension)

o      5 min at 70 °C (enzyme inactivation)

o      hold at 4 °C

·      Set the reaction volume to 20 µl

·      Load the tubes in the cycler and start the run

·      After the run is complete, place the tubes on ice

 

B) Reverse transcription: 2nd strand synthesis

 

Checklist:

cDNA mixture

10X 2nd strand buffer

dNTP mix

Nuclease-free water

E.coli DNA polymerase

RNase H

 

1. Check the 2nd strand buffer for precipitates (undissolved DTT). If precipitates are present, warm the buffer at 37 °C for 5 minutes, then vortex it briefly before using.

 

2. Add the following components to the cDNA mixture on ice:

·      63 ml nuclease-free water

·      10 ml 2nd strand buffer

·      4 ml dNTP mix

·      2 ml DNA polymerase

·      1 ml RNase H

 

 

3. Perform 2nd strand synthesis in the thermal cycler:

·      Program:

o      2 hours at 16 °C (2nd strand synthesis)

o      5 min at 70 °C (enzyme inactivation)

o      hold at 4 °C

·      Set the reaction volume to 100 µl

·      Load the tubes in the cycler and start the run

·      After the run is complete, place the tubes on ice

 

 

C) Purification of cDNA

 

Checklist:

DNA purification column

2 ml receptacle tube

1.5 ml Eppendorf tubes

DNA binding buffer

DNA wash buffer

Nuclease free water

 

1. Mark the DNA purification columns, the receptacle tubes and the 1.5 ml Eppendorf tubes with the RNA code numbers, and place the columns in the 2 ml receptacle tubes.

 

2. Transfer the entire 100 ml 2nd strand synthesis reaction into a new 1.5 ml Eppendorf tube. Add 250 ml DNA binding buffer and vortex briefly to mix.

 

3. cDNA binding to the column matrix

·      Transfer the reaction-DNA binding buffer mixture (350 ml) to the column, then close the column cap.

·      Centrifuge at 10000 g for 1 min.

·      Make sure that the entire volume passed through the column. If it did not, centrifuge it again at 10000 g for 1 min.

·      Remove the column from the tube, discard the flowthrough, then reinsert the column into the tube.

 

4. Wash the cDNA:

·      Add 500 ml DNA wash buffer to the column, then close the column cap.

·      Centrifuge at 10000 g for 1 min.

·      Remove the column from the tube, discard the flowthrough, then reinsert the column into the tube.

·      Centrifuge at 10000 g for 1 min.

 

5. Elute the cDNA: First elution step

·      Transfer the column to a new 1.5 ml elution tube.

·      Pipette 10 ml of nuclease-free water onto the column matrix, then close the tube cap. Make sure the pipette tip does not touch the fiber matrix.

·      Incubate the column at room temperature for 2 min.

·      Centrifuge at 10000 g for 1 min for an elution volume of 9 ml.

 

6. Elute the cDNA: Second elution step

·      Pipette 10 ml of nuclease-free water onto the column matrix, then close the tube cap.

·      Incubate the column at room temperature for 2 min.

·      Centrifuge at 10000 g for 1 min for an elution volume of 18 ml.

 

 

D) Performing in vitro transcription labeling

 

Checklist:

18 ml cDNA output – if less than 18 ml, adjust to 18 ml with nuclease-free water

10X IVT buffer

NTPs

IVT control DNA

IVT enzyme mix: T7 RNA polymerase (75 U/ml), pyrophosphatase (25 U/ml), RNase inhibitor ( 5 U/ml)

DIG-UTP

 

1. Check the IVT buffer mix for precipitates (undissolved DTT). If precipitates are present, warm the buffer at 37 °C for 2-3 minutes, then vortex it briefly before using.

 

2. Transfer 18 ml cDNA output to a new 0.2 ml Eppendorf tube, marked with the sample code. Add the following components at room temperature:

·      4 ml 10X IVT buffer

·      8 ml DIG-UTP

·      4 ml NTP mix

·      2 ml control DNA

·      4 ml IVT enzyme mix

 

Total volume: 40 ml

 

3. Perform IVT in the thermal cycle

·      Program: 9 hours at 37 °C (In vitro transcription)

Indefinite hold at 4 °C

·      Set the reaction volume to 40 µl

·      Load the tubes in the cycler and start the run

·      After the run is complete (next day!!), place the tubes on ice

 

 

 

Practical 3B: Purification and quality control of IVT-labeled cRNA for the microarray experiments

 

 

Introduction :

The product of the IVT reaction is digoxigenin-labeled cRNA. It is important to work carefully with this material to prevent contamination with exogenous RNase. Handle the RNA purification columns by the top edges and do not touch the fiber matrix with the pipette tips.

 

Checklist:

Nuclease-free water

RNA binding buffer

100% ethanol

RNA wash buffer

RNA purification columns

2 ml receptacle tubes

1.5 ml elution tube

 

1. Mark the RNA purification column and receptacle/elution tubes with the sample code numbers. Insert the column in the receptacle tube.

 

2. In a fresh 1.5 ml tube mix the following:

 

 

 

 

3. Centrifuge at 10000 g for 1 min. Make sure that the entire volume passed through the column. If it did not, centrifuge it again at 10000 g for 1 min. Remove the column from the tube, discard the flowthrough, then reinsert the column into the tube.

 

4. Wash the cRNA:

·      Add 650 ml RNA wash buffer to the column, then close the column cap.

·      Centrifuge at 10000 g for 1 min.

·      Remove the column from the tube, discard the flowthrough, then reinsert the column into the tube.

·      Centrifuge at 10000 g for 1 min.

 

 

5. Elute the cRNA (first elution):

·      Transfer the column to a new 1.5 ml elution tube.

·      Pipette 100 ml of nuclease-free water onto the column matrix, then close the tube cap. Make sure the pipette tip does not touch the fiber matrix.

·      Incubate the column at room temperature for 2 min.

·      Centrifuge at 10000 g for 1 min

·      Discard the column then cap the tube.

·      Store the cRNA product on ice while the course staff assesses quantity and quality.

 

6. Quantification of cRNA:

·      Dilute a small amount of cRNA product 1:30 with TE buffer

·      Measure absorbance at 260 nm (Practical 1B)

 

7. Assessing quality: with Agilent 2100 Bioanalyzer, Practical 1C.

 

 

Reagents

 

 

Supplier: Applied Biosystems

 

Chemiluminescent NanoAmp RT-IVT labeling kit

Cat. No. 4365715

 

Microarrays – Human Genome Survey Microarray v2.

Cat. No. 4359029 (4 arrays per package)

 

Chemiluminescent Detection kit

Cat. No. 4339546 and 4341991

 

 

Supplier: Roche

 

DIG-11-UTP, 200 nmol/57 ul

Cat. No. 03359247910

Anti-Digoxigenin-AP, Fab fragments: 150 U

Cat.No.: 1093274