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.
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).
I typically label many samples at a time and use nuclease-free .2 mL strip tubes (VWR 53509-304)
|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)|
(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.
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|
|50X aa-dUPT mix||0.6|
|Superscript II RT||200 U/ul||2|
|50X aa-dUTP mix 1:1 ratio||Concentration||ul|
|aa-dUTP (Ambion 8439)||50 mM||50||store at -20°C in 30ul aliquots|
Incubate at 42°C for 2 hours.
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)
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:
|.22 uM filtered 1M Na2HPO4||98 ul|
|.22 uM filtered 1M NaH2PO4||2 ul|
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.
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.