P-BAIR-2 - P-BAIR-2

Type
labeling
Description
cRNA preparation using phenol

These protocols are based on those provided by Affymetrix and include modifications from the Whitehead Institute for Biomedical Research, MIT, Boston and the CSC/IC Microarray Centre.


A. First strand cDNA synthesis 5.11.01

Things to note:


1.It is very important to start with good quality, clean, total RNA. Acceptable ratios for the absorbance of the total RNA at 260nm and 280nm are between 1.9 and 2.1. Ensure that the total RNA is diluted, so that the absorbances measured are in the linear range for the spectrophotometer. This is often between 0.1 and 0.4 absorbance units.

2.For step (1) we recommend starting with 10µg total RNA per reaction tube. Do not start with less than 10µg total RNA per reaction. Carry out two reactions for each sample if you require more than 40µg of cRNA.

3.Steps (3) and (4) are key to the preparation of cDNA. Step (3) breaks any secondary structures of the RNA while step (4) inhibits the reoccurrence of secondary structures.

4.Make a 'master mix' for step (5). Mix thoroughly and spin briefly, as necessary.

5.Use RNase free microfuge tubes and pipette tips throughout.


Method

1. Place the following in a microfuge tube:

-µl (10µg) total RNA in nuclease free treated dH2O

-µl (100 pmol) T7-(T)24 primer

(GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24)

1µl Poly A+ Controls

-µl nuclease free dH2O

11 µl final volume

2.Mix by pipetting, spin briefly as necessary.

3.Incubate at 65-70oC for 10 minutes.

4.Place tubes on ice.

5.Prepare a master mix on ice. The quantities are for each reaction/tube:

4µl (5x) First Strand Buffer (thaw at 37oC, put on ice), Invitrogen Y02321

2µl (0.1 M) DTT, Invitrogen Y00147

1µl (10mM) dNTPs, Invitrogen 18427-013

7µl

Mix by pipetting, spin briefly as necessary.Add 7µl of master mix to each reaction tube.

6.Incubate at 42 oC for 2 minutes.

7.Add 2µl Superscript II reverse transcriptase (400 U total), Invitrogen 18064-014.

8.Tap tube to mix, spin briefly as necessary.

9.Incubate at 42oC for 1 hour in a waterbath.

10.Place tube on ice and proceed to 'Second strand cDNA synthesis', or transfer tube to dry ice, allow reaction to freeze and store at -80oC.


B. Second strand cDNA synthesis 12.02.01.


Things to note:

1.Check the units/µl for the RNase H, it is important that exactly 2U are used per reaction; you may have to adjust volumes accordingly.

2.Make a 'master mix' for step (2). Mix thoroughly because the enzymes are in glycerol. Spin briefly, as necessary.

3.For step (4), incubate either in a water bath in the cold room or in a refrigerated block with a lid, to reduce condensation.

4.For step (5), store at -80oC only if you cannot go to the 'Clean-up of double stranded cDNA' step immediately after the second strand cDNA synthesis is completed.


Method

1.Place all reagents and first strand reaction tubes on ice. Assemble master mix on ice.

2.Prepare a master mix in a new microfuge tube. For each reaction use:

-µl nuclease free treated dH2O

30µl (5x) Second Strand Buffer, Invitrogen 10812-014

3µl (10mM) dNTPs, Invitrogen 18427-013

1µl DNA Ligase (10 Units), Invitrogen 18052-019

4µl DNA Pol I (40 Units), Invitrogen 18010-025

-µl RNase H (2 Units), Invitrogen 18021-071

130µl

Mix by vortexing, spin briefly as necessary. Add 130µl of the master mix to each of the first strand reaction tubes.

3.Mix by pipetting, spin briefly as necessary.

4.Incubate at 16oC for 2 hours. Use a water bath in a cold room or a refrigerated block with lid, if possible. This reduces condensation.

5.Place tube on ice and proceed to 'Clean-up of double stranded cDNA', or transfer tube to dry ice, freeze and store at -80oC.


C. Clean-up of double stranded cDNA 25.09.02.


Things to note:

1. Double stranded cRNA clean up is carried out using the GeneChipý Sample Cleanup Module, Affymetrix 900371. Prepare reagents following the instructions in the kit.

2. Perform the clean up in a 1.5 or 2ml microfuge tube.

3. Ensure ethanol has been added to cDNA Wash Buffer before use.

4. After the clean up, ALL of the double stranded cDNA is used for the in vitro transcription reaction. Do not attempt to quantify the products of the reaction.


Method

1. Add 600µl cDNA Binding Buffer to the cDNA reaction. Mix by pipetting.

2. Check that the colour of the mixture is yellow (If the colour is orange or violet, add 10µl of 3 M sodium acetate, pH 5.0 and mix).

3. Apply 500µl of the sample to the cDNA Cleanup Spin Column.

4. Centrifuge at >=8 000g (>= 10 000rpm) for 1 minute.

5. Empty the collection tube.

6. Reload the spin column with the remaining mixture.

7. Centrifuge at >=8,000g (>= 10,000rpm) for 1 minute.

8. Transfer the spin column into a new 2ml Collection tube.

9. Pipette 750µl cDNA Wash Buffer onto the spin column.

10.Centrifuge at >=8 000g (>= 10 000rpm) for 1 minute.

11.Empty the collection tube.

12.Open the cap of the spin column and centrifuge at maximum speed for 5 minutes, to completely dry the membrane.

13.Transfer the spin column to a fresh 1.5ml collection tube.

14.Elute by placing 14µl of cDNA Elution Buffer directly onto the middle of the spin column membrane.

15.Incubate at room temperature for 1 minute.

16.Centrifuge at maximum speed for 1 minute.

17.Place tube on dry ice to freeze and then store at-80oC or proceed directly to the in vitro transcription reaction.


D. Preparation of cRNA 07.03.03


Things to note:

1.Once reagents have thawed, keep them at room temperature until incubation at 37oC to reduce precipitation of DTT.

2.The incubation should be carried out in an incubator or warm-room, to reduce condensation on the inside of the lid.

3.The reagents used for in vitro transcription are from the Bioarray High Yield RNA Transcript Labelling Kit, Enzo 900182.


Method:

1.Thaw all reagents and double stranded cDNA at room temperature.

2.Prepare a master mix of the IVT reagents at room temperature. The quantities are for each reaction/tube:

12µl double stranded cDNA from previous method.

10µl nuclease free dH20

4µl (10x) HY Reaction Buffer

4µl Biotin-labelled ribonucleotides

4µl DTT

4µl RNase Inhibitor Mix

2µl T7 RNA Polymerase

40µl

3.Mix by pipetting, spin briefly as necessary.

4.Incubate at 37oC for 5 hours. Gently mix the reaction every hour by tapping tube, spin briefly as necessary.

5.Proceed to 'Clean-up of cRNA', or freeze by placing tube on dry ice and store at -80oC.


E. Clean-up of cRNA 07.03.03.


Things to note:

1.Removal of unincorporated biotinylated ribonucleotides is very important

obtaining a good quality scan.

2. cRNA clean up is carried out using the GeneChip Sample Cleanup Module,Affymetrix 900371. Prepare reagents following the instructions in the kit.

3. Ensure ethanol has been added to IVT cRNA Wash Buffer before use.

4. Be careful not to touch the tip of the spin column at any time.

5. Carry out centrifugation at >= 8,000g (>= 10,000rpm).


Method:

1.Add to the in vitro transcription reaction tubes:

60µl nuclease free dH2O

350µl IVT cRNA Binding Buffer

2.Mix thoroughly by pipetting.

3.Add 250µl (100%) ethanol. Mix by pipetting. Do not spin.

4.Apply sample to an IVT cRNA Cleanup Spin Column.

5.Spin at >= 8,000g (>= 10,000rpm) for 15 seconds.

6.Transfer the spin column to a fresh 2ml collection tube.

7.Add 500µl IVT cRNA Wash Buffer onto the spin column.

8.Spin at >= 8,000g (>= 10,000rpm) for 15 seconds.

9.Empty the collection tube.

10.Add 500µl (80%) ethanol onto the spin column.

11.Spin at >= 8,000g (>= 10,000rpm) for 15 seconds.

12.Empty the collection tube.

13.Open the cap of the spin column and centrifuge at maximum speed for 5 minutes to completely dry the membrane.

14.Transfer spin column into a new 1.5ml Collection tube.

15.Elute by placing 11µl of RNase-free water directly onto the middle of the spin column membrane.

16.Incubate at room temperature for 2 minutes.

17.Spin at maximum speed for 1 minute.

18.Add 10µl of RNase-free water directly onto the middle of the spin column membrane.

19.Incubate at room temperature for 2 minutes.

20.Spin at maximum speed for 1 minute.

21.Dilute an aliquot of the eluate with dH2O (not DEPC treated) and measure the absorbance at 260nm and 280nm. The dilution should be sufficient so that readings obtained are in the linear range for the spectrophotometer. Unless known otherwise assume this is between 0.1 and 0.4 absorbance units (try preparing a serial dilution, diluting 2µl of eluate in 48µl of dH2O and then this 50µl in 450µl of dH2O). The cRNA should be clean e.g. a ratio of the absorbances at 260nm to 280nm should be around 2.0, with an acceptable range of 1.9 to 2.1. You should have 40 to 110µg of cRNA (assuming that you started with 10µg total RNA where 1 OD260 unit is equivalent to 40µg/ml single stranded RNA). If you have a yield of less than 40µg of cRNA per reaction (per 10µg reaction of total RNA), we recommend that you do not use the cRNA, as there is likely to be a problem with sample preparation.

22.Run a sample of the total RNA and cRNA for each sample (500ng to 1µg) on a denaturing 1% agarose gel along with a marker spanning 200bp to 3kb (a DNA marker gives a rough idea of the size). The cRNA product should be a smear from about 100bp to 2kb with a brighter region from 500bp to 1kb, there is an example image for guidance at the end of this protocol. Photograph the gel, so that the markers are easy to identify and label the lanes with the sample identifier.

23.Freeze by placing tube on dry ice and store at 80 °C.

24.Bring the cRNA to the Microarray Centre on dry ice (after prior consultation with Helen Banks or Nicola Cooley to arrange a convenient time). Note that each sample should be clearly labelled on both the top and side of each tube with the date (day, month, year e.g. 05.11.01) and the sample identifier.

In order to assess the quality of the cRNA samples, prior to hybridisation on test arrays, we also require the following information, for both the cRNA samples and the total RNA from which it was generated.

(a) the raw absorbance readings

(b) the ratio of the absorbances at 260nm and 280nm.

(c) the cRNA yield from 10µg of total RNA.

(d) an original photograph of the gel, with the markers and samples labelled.

Links
All 13 experiments using protocol P-BAIR-2