E.coli Total RNA Labeling Protocol for High Density
Oligonucleotide Array
(updated/corrected 5-30-2007)

RNA Preparation

• If RNA is in ethanol, spin down 10 ug of RNA per reaction @ 14000 rpm for 20 min at 4°C.
• Pipette off supernatant and wash pellet with 100 ul of 70% ETOH. (prepared with DEPC H₂O)
• Spin 5 min and remove supernatant without disturbing pellet
• Air dry pellet 8-10 min at Room Temp (RT). (Caution: if pellet is over dried it is hard to resuspend!)
• Resuspend pellet in 11 ul DEPC H₂O and add 1 ul 0.5 ug/ul Random Hexamer; this is the RNA/Primer Mix.
• Heat to 70°C for 5 min and chill on ice for 2 min, quick spin.
• Mix following reagents:

RNA/Primer Mix		12 ul
dNTP Mix	10mM	3 ul
First-Strand Buffer	5x	12 ul
DTT	0.1M	3 ul
DEPC H2O		24 ul
SuperScript II	200 U/ul	6 ul
Total		60 ul

• Mix everything except Superscript II. Heat to 42°C for 1 min, then add enzyme. (Can make the mix and add 42 ul mix to RNA/Primer mix)
• Incubate 90 min at 42°C.
• Inactivate the reaction by heating at 70°C for 15 min then chill on ice

Digest RNA and Purification of cDNA

RT reaction		60 ul
RNase H	2 U/ul	1 ul
RNase A	1 ug/ul	1 ul
H2O		38 ul
Total		100 ul

• Incubate 10 min at 37°C.

Purify cDNA with Qiaquick PCR purification kit

• Add 500 ul of Buffer PB to 100 ul sample.
• Place a QIAquick spin column in a provided 2 ml collection tube.
• Apply the sample to the QIAquick column and centrifuge for 1 min
• Discard flow-through. Place the column back into the same tube.
• Add 750 ul Buffer PE to the column and spin for 1 min.
• Discard flow-through and place the column back in the same tube. Spin for additional 1 min.
• Place the column in a clean 1.5 ml microcentrifuge tube.
• Add 34 ul Buffer EB (10mM Tris.Cl, pH 8.5) or H₂O to the center of the QIAquick membrane, let the column stand for 1 min, and centrifuge the column for 1 min. (Finally volume is about 32 ul)
• Quantitate cDNA in spectrophotometer with 1 ul sample (1 A₂₆₀ = 0.033 ug/ul), save another 1 ul for gel.

cDNA Fragmentation and end labeling

• Dilute 1 U/ul Dnase I to 0.1 U/ul with 1x Dnase I Buffer.
• Mix following reagents:

cDNA		30 ul
DNase I Buffer	10x	4 ul
H2O		4 ul
DNase I	0.1 U/ul	2 ul
Total		40 ul

• Incubate 10 min at 37°C*
• Heat to 99°C for 10 min to stop the reaction
• Check fragmentation on 2% agarose gel, (normally use 2 ul) avg. length 50-100 bp

* incubation time may need to be adjusted for different batches of DNase I due to varying activity.

Labeling with Terminal Transferase

• Mix following together

Fragmented cDNA		38 ul
TdT Buffer (NEB Buffer 4)	10x	10 ul
CoCl2	2.5mM	10 ul
Biotin-11-ddATP**	1mM	2.5 ul
H2O		37 ul
TdT	20 U/ul	2.5 ul
Total		100 ul

• Incubate 2 hours at 37°C
• Optional: Check fragmentation on 2% agarose gel with gel shift assay
• Freeze at -20°C, ready to mix with hybridization cocktail

Reagents and Suppliers

Biotin-11-ddATP**		PerkinElmer	NEL508
SuperScript II	200 U/ul	Invitrogen	18064-014
dNTP set, 100mM solutions		Amersham	27-2035-01
pd(N)6 Random Hexamer***	50 A260 U	Amersham	27-2166-01
QIAquick PCR Purification Kit		Qiagen

** this protocol originally specified Biotin-N⁶-ddATP (PerkinElmer EL508), which is no longer available; according to PerkinElmer, Biotin-11-ddATP can be directly substituted

*** supplied as a lyophilized sodium salt with 5' phosphorylated ends; resuspend at desired concentration

Note: This protocol was adapted from the original Affymetrix protocol.