Evaluation of theTrizol Method vs Qiagen AllPrep

for the Extraction ofDNA, RNA and Protein

(last updated on 3/18/2008)

Objective

• To test the suitability of the Qiagen AllPrep method as an alternative tothe Trizol method for the extraction of DNA, RNA and protein fromPBLs

• To have the ability to obtain all three fractions from PBLs using a singleprotocol reducing sample to sample variability and improve efficiencyfrom valuable, limited clinical samples

Background

• Currently we use the Trizol (Invitrogen) method, a labor intensive protocol that can easily take over a dayto process a couple of samples for DNA, RNA and protein extraction

• Qiagen AllPrep is a more simplified protocol, takes less time, avoids the use of toxic phenol andchloroform, does not require ETOH precipitation and is easier to process multiple samples simultaneously

• The major concern for us was whether the DNA, RNA and protein derived from the AllPrep method wouldbe able to produce downstream data over multiple platforms that was acceptable if not superior to thecurrent method, that is, genomic DNA for SNP genotyping using the Affymetrix 6.0 array, total RNA foramplification/labeling and hybridization to Affymetrix GeneChip arrays and protein suitable for tandemmass spectrometry

• PBLs were isolated from 8 mls of whole blood collected in Cell Preparation Tubes (CPT; BectonDickenson) drawn from normal blood donors

Trizol Method

DNA redissolve

DNA wash

DNA precipitation

~2.5 hours

RNA precipitation

RNA wash

Qiagen RNeasy clean up

RNA redissolve

~2 hours

Protein redissolve

Protein precipitation

Protein wash

~3 hours

phase separation

Cells or Tissue

lyse / homogenize

~1 hour(depending onsample type)

(needle/syringe for cells, glass/ teflon homogenizer for tissues)

~1 hour

~1 hour(depending onsample type)

DNATrizol vs AllPrep

Donor 1 PBLs(D1)

Donor 2 PBLs(D2)

A260/280 Yield (ug) A260/280 Yield (ug)

Trizol 1.61 12.4 1.79 20.4

AllPrep 1.8 3.9 1.93 12.6

PBLs derived from a single 8 ml draw CPT tube for each method

23kb>

D1 D2 1ug 1ug

Trizol

D1 D2 250ng 1ug

AllPrepDNA yields were derived from 8mls of whole blood. Thedifference in the band size between the Trizol and the AllPrepmethod is due to the different protocols. Average length forthe AllPrep method is typically 15-30kb.

SizeMarker

SizeMarker

Affymetrix SNP Array 6.0 Results

ExtractionMethod

CallRate

QC CallRate

ComputedGender

QC CallRate(Nsp)

QC Call Rate(Nsp/StyOverlap)

QC CallRate(Sty)

GenderMatch?

Donor 1PBLs

Trizol 94.17 92.65 male 90.87 94.64 89.69 yes

AllPrep 99.27 98.41 male 97.69 99.08 97.58 yes

Donor 2PBLs

Trizol 97.5 95.76 male 93.57 97.47 94.04 yes

AllPrep 99.12 97.49 male 96.92 97.97 96.94yes

QC Call Rates for both Trizol and AllPrep passed the standardcutoff of 86% but AllPrep had much higher call rates

RNATrizol vs AllPrep

Donor 1 PBLs Donor 2 PBLs

A260/280 Yield (ug) A260/280 Yield (ug)

Trizol 2.01 4.5 2.06 10.4

AllPrep 2.03 3.7 2.04 10.5

PBLs derived from a single 8 ml draw CPT tube for each method

RNA yields between Trizol and AllPrep were similar and whatwould be expected from 8 mls of whole blood

Agilent BioAnalyzer Results

• RNA derived using the AllPrep method subsequently labeled and hybridized to GeneChips, data to follow

Donor 1 Trizol

Donor 2 AllPrepDonor 1 AllPrep

Donor 2 Trizol

Agilent BioAnalyzer traces show high quality RNA wasderived from both Trizol and AllPrep methods

Labeling of RNA

• Used Ambion MessageAmp II Biotin Enhanced Kit• 1 ug total RNA starting amount

ExtractionMethod A260/280 aRNA yield

(ug)

Donor 1 PBLs AllPrep 2.20 65.4

Donor 2 PBLs AllPrep 2.24 89.2

High aRNA yields for both donors(Trizol method RNA samples were notprocessed further)

Affymetrix Hu133A Plus 2.0 GeneChipQC Results

ExtractionMethod

Background Noise %Present

GAPDH3’/5’

Actin3’/5

SpikedControls

ScaleFactor

Donor 1PBLs

AllPrep 37.64 1.59 56.8 0.95 0.91 yes 1.693

Donor 2PBLs

AllPrep 37.23 1.55 57.6 1.02 1.02 yes 1.642

Both donors passed all standard QC parameters

ProteinYield and Tandem MS Results

Average protein yields and number of proteins identified for the Trizol and AllPrep methods are shown above.Both methods are compatible with tandem MS analysis although yields were lower for the AllPrep method.

Avg Protein Ids *

Protein yield with AllPrep method(derived from 8mls of blood)

Sample 1 50 ug 1258

Sample 2 90 ug 1119

Protein yield with Trizol method(derived from 8mls of blood)

Sample 3 170 ug1224**

Sample 4 180 ug

** Pooled 50ug of Sample 3 and Sample 4

* at 5% peptide FDR

Summary

• DNA derived from PBLs using AllPrep generated improved AffymetrixSNP 6.0 results compared to the Trizol method seen as an increase in theQC Call Rate

• Total RNA derived from PBLs using AllPrep provided RNA of almostidentical yield and of high quality compared to the Trizol methodgenerating gene expression data passing all the standard AffymetrixGeneChip QC parameters

• Protein derived from PBLs using AllPrep provided excellent tandem MSproteomics results

• We obtained lower yields of DNA and protein using the AllPrep method.According to the Qiagen Manual, cell disruption and homogenizationsteps can be optimized to improve yields for DNA and protein.

Conclusion

• Qiagen’s AllPrep DNA/RNA/Protein protocol requires less than a quarter of thetime than the Trizol method saving us considerable labor costs.

• The use of the AllPrep method eliminates the use of toxic phenol/chloroform -basedchemicals in the lab

• Comparable data was generated from AllPrep derived specimens. AllPrep is asuitable replacement to the Trizol method for extracting DNA, RNA and proteinfrom PBLs.