Reverse Transcription (RT) and Amino-Allyl coupling of cDNA for (total) RNA sample input

Jason Myers, Kate Rubins and Dan Hogan

DRAFT VERSION 01.25.06

Note

This is a (slightly) modified version of a protocol from Joe Derisi's lab at the 2003 UCSF Microarray course. This modified protocol was developed by Jason Myers, Kate Rubins and Dan Hogan and has room for refinement.

1. RT Reaction

For each array, you will need to set up two RT reactions, one for your Cy5 reaction (often experimental) and one for your Cy3 reaction (often reference).

A. sample RNA, doping control RNA, primer preparation

I typically label many samples at a time and use nuclease-free .2 mL strip tubes (VWR 53509-304)

Priming Reaction Concentration/Amount ul
Oligo dT20V (note 1) 5 ug/ul 1
Doping control (note 2) 1
Rnase inhibitor (note 3) 10U 0.25-5
Sample (total) RNA up to 50 ug
Rnase-free H2O (note 4)
Final 18.4

(Note 1)Depending on your sample, you may want to use dT20V/N9 (5 ug/ul each). We purchase our primers from Invitrogen and have them HPLC purified.
(Note 2)Doping controls are available through Stanford Microarray Core Facility and come with instructions. Remember there are different doping control mixes for Cy5 and Cy3 samples.
(Note 3)This is optional. Commercially available Rnase inhibitors only inhibit a subset of Rnases e.g. A, B, C, T1, and 1.
(Note 4)We use non-DEPC treated nuclease free water from Ambion (9932).

Incubate samples at 70°C for 10 min.
Remove and immediately place on ice for 10 min.

B. cDNA synthesis

Add 11.6 ul of the cocktail below to the RNA sample on ice or in cold room to yield final reaction volume of 30 ul.

Cocktail concentration ul per reaction
5X buffer 6
50X aa-dUPT mix 0.6
DTT/td> 0.1M 3
Superscript II RT 200 U/ul 2

50X aa-dUTP mix 1:1 ratio Concentration ul
dATP 100 mM 50
dCTP 100 mM 50
dGTP 100 mM 50
dTTP 100 mM 25
aa-dUTP (Ambion 8439) 50 mM 50
store at -20°C in 30ul aliquots

Incubate at 42°C for 2 hours.

2. Hydrolysis

Incubate at 95°C for 5 minutes (samples can be stored at 4°C or -20°C)
Add 13 ul 1N NaOH, 1ul .5M EDTA (pH is now ~13)
Incubate at 67°C for 15 minutes
Neutralize with 50ul 1M Hepes pH 7.0 (pH is now ~7.5) (samples can be stored at 4°C or -20°C)

3. Cleanup

The purpose of the cleanup is to remove everything but the cDNA.
NOTE: It is crucial to remove all free amine groups before coupling to NHS-ester dyes so DO NOT elute with Tris containing buffer.
We use Qiagen Minelute Reaction Cleanup (28206).

To prepare 10mM Na-Phosphate solution pH 8.5:

volume
.22 uM filtered 1M Na2HPO4 98 ul
.22 uM filtered 1M NaH2PO4 2 ul
water 9.9 ml

Note: many protocols suggest using 50-100mM Na-Bicarbonate pH 9.0 for coupling, but it is optimal to elute from the columns at pH 7-8.5, I find Na-Phosphate easier to work with than Na-Bicarbonate, and I see no difference in coupling efficiency between the two conditions.

4. Coupling

In the coupling reaction the terminal amino group on the aa-dUTP attacks the NHS-monoester dye to form a Cy-aa-UTP covalent bond. The reactions are performed at pH 8.5-9, which is an optimal balance between deprotonation of the amino group and nucleophilic attack and hydrolysis reactions of the NHS ester with water. The dyes should not be exposed to water prior to coupling and should be used immediately after they are resuspended in DMSO. Reactions should be carried out in the dark as the Cy dyes are light sensitive.

Note: Depending on the concentration of cDNA, you may be able to use one dye pack for several cDNA samples. It is worth testing coupling efficiency with sample cDNA as cutting down on the number of dye packs will save lots of money over time (~$10/pack). If you use one dye pack for multiple samples, it is important to maintain 50% v/v DMSO in the coupling reaction.

5. Removal of unincorporated dyes

To remove unincorporated dyes I use the Qiagen Minelute. NOTE: (Optional) Some protocols call for quenching the coupling reaction by addition of 4.5 ul 3M hydroxylamine and incubation for 15 min at RT in dark before proceeding to cleanup. You can pool the samples and use half as many columns. I skip this step as addition of ERC brings pH of solution down to ~6.6 and should effectively quench the reaction. However, I have not compared the methods head-to-head.