cDNA Synthesis and Alexa dye labeling in Preparation for Microarray hybridization

1)      Denaturation of RNA and annealing of primers:

            15 µg total RNA

            random 18-mer at 2 µg/µl, 4 µg=2 µl

            random hexamers at 3 µg/ µl, 10 µg=3.3 µl

            RNase-free ddw QS to 18 µl

Or

            isolated mRNA from 10 µg of total RNA

            random hexamers at 3 µg/ µl, 5 µg=1.6µl

            RNase-free ddw QS to 18 µl

Incubate each tube at 70^oC for 5 min. to denature and then on ice for 1 minor more.  Note that it is important to use the random 18-mer in addition to the random hexamers when starting with total RNA in order to favor priming of mRNA in the reaction.  Also, I have found that the primer/RNA ratio stated above yields the highest amount of cDNA.

2)      cDNA synthesis with amino-modified nucleotides:

            With samples on ice, add to each tube:

5X first strand buffer                  6.0 µl

0.1M DTT                                1.5 µl

dNTP mix                                 1.5 µl

RNase out                                 1.0 µl

Superscript RT              2.0 µl

Final volume                              30 µl

Mix well, centrifuge briefly to collect contents at the bottom of the tube.  Incubate tubes at 46^oC for 2-3 hours.  3 hour incubations will result in 20-30% higher cDNA yields.

3)      Alkaline Hydrolysis of RNA and Neutralization:

Add 15 µl of 1N NaOH to each reaction tube.  Mix thoroughly

Incubate at 70^oC for 10 min

Neutralize with 15 µl of 1N HCL, mix gently

4)      Purification of cDNA using Qiagen PCR purification kit

Add 300 µl of buffer PB to sample, mix, load onto Qiaquick column

Centrifuge at room temperature for 1 min

Discard flow through

Add 750 µl of wash buffer PE (make sure you ve added ethanol to the buffer)

Centrifuge at room temperature for 1 min

Discard flow through

Centrifuge for another min. to dry out the column

Move column to a new eppendorf tube

Apply 30 µl RNase-free sterile ddw to the column membrane

Allow to sit at room temperature for 1 min.

Centrifuge for 1 min to collect the cDNA

Quantitate the yield of cDNA by diluting 2 µl (+98 µl ddw), read A₂₆₀

Typical yields of cDNA starting from total RNA are around 10 µg and from mRNA-around 1 µg or more.

5)      Coupling with Alexa dyes:

Dry cDNA in speedvac on medium heat setting until the sample is in 3 µl or less, DO NOT dry completely or the cDNA will be stuck to the tube and be very hard to resuspend (check every 5 min. or less).

Add 5 µl 2X coupling buffer to each sample

Add 2 µl room temperature DMSO to a vial of Alex Fluor to resuspend dye

Add DMSO/dye solution to cDNA

Incubate at room temperature in the dark for 1-2 hours

Reaction can be stored overnight in the dark if needed

6)      Purification of Fluorescently labeled cDNA using Qiagen PCR purification kit

Add 30 µl of buffer PB to sample, mix, load onto Qiaquick column

Centrifuge at room temperature for 1 min

Discard flow through

Add 750 µl of wash buffer PE (make sure you ve added ethanol to the buffer)

Centrifuge at room temperature for 1 min

Discard flow through, there should be color on the column at this point

Centrifuge for another min. to dry out the column

Move column to a new eppendorf tube

Apply 30 µl RNase-free sterile ddw to the column membrane

Allow to sit at room temperature for 1 min.

Centrifuge for 1 min to collect the dye labeled cDNA, these should be vibrantly colored

Analyze 2 µl (+98 µl ddw) on the spec to determine yield

Scan samples from 240-800 nm and calculate the following:

ng of total cDNA=(A₂₆₀-A₃₂₀) X 37ng/ml X volume in ml X dilution factor

pmoles Alexa Fluor 555=(A₅₅₅-A₆₅₀)/0.15 X volume in ml X dilution factor

pmoles Alexa Fluor 647=(A₆₅₀-A₇₅₀)/0.24 X volume in ml X dilution factor

Base/dye ratio for Fluor 555={(A₂₆₀-A₃₂₀)-[(A₅₅₅-A₆₅₀) X 0.04]} X 150,000/(A₅₅₅-A₆₅₀) X 6,600

Base/dye ratio for Fluor 647={(A₂₆₀-A₃₂₀)-[(A₆₅₀-A₇₅₀) X 0]} X 239,000/(A₆₅₀-A₇₅₀) X 6,600

The number of dye molecules per 100 bases is calculated:

100/(base/dye ratio)

You can minimally expect >24 pmole of incorporated dye and >2.5 dye molecules/100 bases.  I typically get around 900-1000 pmoles of dye incorporation when starting with total RNA and around 150 pmoles of dye incorporation for mRNA and 3.5-5 molecules of dye/100 bases.

Typically the cDNA from total RNA averages 500 bp in length as determined by gel electrophoresis with a range of ~1kb to ~200 bp relative to 100bp standard ladder.

Random Hexamers=Invitrogen cat# 48190-011, 3 µg/ul

Tandom 18-mer=Invitrogen custom oligo synthesis 50 nM synthesis $7.20, quote #508786

Cautions:

The DMSO needs to be at room temperature before you open the tube so that it does not absorb moisture from the air which will prevent it from working properly.

When adding ddw to the Qiagen membrane for elution, make sure the water is added to the surface of the membrane and not caught up on the sides of the column, otherwise the yield will be decreased.

It is important to plan your experiment such that you know what array hybridizations you will be performing at the same time and what samples are to be compared directly so that you will know what RNAs and cDNAs will need to be prepared at the same time.  It will not be possible to compare samples prepped on different days

.

You should have minimally 50 pmoles of each dye in an array hyb.

Always check the yield of cDNA in each reaction before proceeding with the dye labeling reaction.  If you do not have the expected cDNA yield you will need to trouble shoot the first part of this procedure.