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

Note:
Start with 10 ug of total RNA for each labeling reaction.
All solutions that can be filtered should be filtered.

RNA Preparation

  • If RNA is in ethanol, spin down 10 ug of RNA per reaction @ 14000 rpm for 20 min at 4C.
  • 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 70C 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 42C for 1 min, then add enzyme. (Can make the mix and add 42 ul mix to RNA/Primer mix)
  • Incubate 90 min at 42C.
  • Inactivate the reaction by heating at 70C 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 37C.

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 37C*
  • Heat to 99C 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 37C
  • Optional: Check fragmentation on 2% agarose gel with gel shift assay
  • Freeze at -20C, 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.


© 2002-2014 UW E. coli Genome Project