Download Simultanous isolation of RNA and DNA from one FFPE

Simultaneous isolation of RNA and DNA from
one FFPE sample
Stefanie Schröer, Christiane Bäumer, Vera Holländer
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
Genomic DNA: Yield and fragment length
Introduction
Worldwide, there are millions of tissue samples archived in tissue biobanks and
biorepositories. These samples are extremely valuable for pharmacological
and biomedical research and companion diagnostics, due to the linkage to
patient history. The vast majority of archived tissue samples are formalin-fixed
and paraffin-embedded (FFPE), since formalin is the standard fixative for
tissue samples.
AllPrep DNA/RNA FFPE Procedure
FFPE tissue sections
Remove paraffin and
dry, then lyse with
proteinase K digestion
Cool on ice and then centrifuge
to obtain RNA-containing
supernatant and
DNA-containing pellet
FFPE blocks serve as an excellent source for histomorphology studies, but their
use in molecular studies is challenging, due to crosslinking and fragmentation
caused by fixation, processing, embedding, and storage conditions.
Since FFPE samples contain DNA molecules that are crosslinked to each other, as well as to RNA and protein
molecules, breakage of these crosslinks is necessary in order to release DNA for subsequent purification. After
differential solubilization, RNA is removed with the supernatant and DNA remains in an insoluble pellet, which is
then further lysed. Chemical modifications due to crosslinking are reversed by subsequent incubation, as chemically
modified DNA is less efficiently recovered during the purification procedure and represents a poor substrate for PCR
and other enzymatic assays.
A
Incubate
supernatant
at 80°C
For reliable comparison of genomic and transcriptomic data from heterogeneous
samples and to spare sample material, purification of DNA and RNA from
the same sample is essential. This is particularly important when working with
tumorous tissues, which contain a heterogeneous distribution of healthy and
malignant cells.
Lyse pellet with
proteinase K
digestion, then
incubate at 90°C
B
20
18
Spleen
A
AllPrep FFPE
QIAamp FFPE
Heart
C
A
Lung
C
A
C
M
16
Bind genomic
DNA
Total RNA
Genomic DNA
The AllPrep DNA/RNA FFPE Kit is designed to simultaneously purifiy
genomic DNA and total RNA from FFPE tissue sections. FFPE samples are
incubated in an optimized lysis buffer, resulting in the release of RNA and
precipitation of DNA. After centrifugation, the RNA-containing supernatant
and DNA-containing pellet are processed separately to purify RNA and DNA.
12
10
8
Treat with
DNase, then
wash
®
14
DNA (µg)
Bind total RNA
Wash
6
4
2
Elute
Elute
Eluted RNA
Eluted DNA
0
Lung
(6 months)
Heart
(13 months)
Spleen
(19 months)
Genomic DNA purified from various FFPE rat tissues stored at room temperature for the times indicated. Purification was performed using either the AllPrep DNA/RNA FFPE Kit
A DNA yields from 20 µm sections were determined by OD measurement.
or, as a control, the QIAamp® FFPE Tissue Kit including RNAse digestion during sample preparation. ■
B Agarose gel analysis of the same volume of eluates. A: AllPrep DNA/RNA FFPE Kit; C: Control; QIAamp FFPE Tissue Kit. M: Lambda Hind III marker.
■
RNA: Yield and integrity
Genomic DNA: PCR analysis and Pyrosequencing®
During differential solubilization, RNA is released and subjected to reverse crosslinking using optimized conditions
that avoid additional fragmentation. This yields high-quality RNA, whilst maintaining RNA integrity for demanding
downstream applications.
The AllPrep DNA/RNA FFPE Kit yields DNA suitable as template for downstream applications, such as real-time PCR
or Pyrosequencing. Pyrosequencing is a technology that allows reliable sequencing of nucleotide sequences, plus
sensitive, accurate quantification of genetic variations within the sequences of interest, such as methylation at CpG
sites or mutations.
B
nt
14
AllPrep
RNeasy
Total RNA was purified from various rat FFPE tissues,
stored at room temperature for the durations shown,
using either the AllPrep DNA/RNA FFPE Kit or the
miRNeasy FFPE Kit. nt: Nucleotide.
AllPrep FFPE
RNeasy FFPE
12
RNA (µg)
10
A
■
6
RNA yield from 1 (spleen, liver) or 2 10 µm
sections (heart, lung) per sample determined by
OD measurement.
4
B The same volume of RNA purified from 1 x 10 µm
■
8
section of rat kidney was analyzed on an Agilent
2100 Bioanalyzer.
2
0
Heart
(4 months)
Lung
(4 months)
Liver
(4 months)
A
B
35
30
CT value
A
Spleen
(29 months)
KRAS Codon 12/13
WT: GGTGGC
AllPrep FFPE
QIAamp FFPE
25
20
15
10
Kidney
(3 days)
Heart
(3 days)
Liver
(19 months)
Lung
(23 months)
60
50
40
30
20
10
0
Total RNA was purified from human breast FFPE
tissue using the AllPrep DNA/RNA FFPE Kit. RNA
was reverse-transcribed using RT2 FFPE PreAMP
technology. Gene expression analysis by real-time
PCR was performed using the Human Cell Cycle RT2
Profiler PCR Array, comparing a tumor and non-tumor
sample. ΔΔCT analysis shows the x-fold difference in
gene expression of the tumor sample compared with
the non-tumor sample.
ABL1
BAX
BCL2
CCNB1
CCNC
CCND1
CCND2
CCNG1
CCNG2
CCNH
CCNT2
CDC16
CDC2
CDK2
CDK4
CDK6
CDK7
CDK8
CDKN1A
CDKN1B
CDKN3
CHEK1
CHEK2
CKS2
CUL1
CUL2
CUL3
DDX1
GTF2H1
HERC5
HUS1
KNTC1
MAD2L1
MCM3
MCM4
MCM5
MKI67
MNAT1
RAD1
RAD17
x-fold difference in
gene expression
RNA: RT2 FFPE PreAmp technology
A
■
KRAS Codon 61
WT: CAA, reverse
sequenced as TTG
DNA was purified from various FFPE rat tissues stored at room temperature, as indicated, using either the AllPrep DNA/RNA FFPE Kit or, as a control, the QIAamp FFPE
Tissue Kit. Real-time PCR was carried out using the QuantiTect® SYBR Green PCR Kit to analyze a 78 bp amplicon of the Prnp gene.
B DNA was purified from breast cancer FFPE tissue using the AllPrep DNA/RNA FFPE Tissue Kit. Pyrosequencing to identify mutation of the KRAS gene was performed using
■
the therascreen KRAS Pyro Kit.
RNA: Real-time RT-PCR
Conclusions
Genomic DNA contamination in an RNA sample affects the accuracy of gene expression analysis by real-time RT-PCR
if the primers used amplify both cDNA and gDNA sequences. Therefore, elimination of gDNA contamination is
essential for accurate results. This can be achieved by purifying RNA using the AllPrep DNA/RNA FFPE Kit. Depending
on the RNA binding conditions, small RNAs (such as microRNA) are either present or absent in the purified RNA.
The AllPrep DNA/RNA FFPE Kit provides:
■ Simultaneous purification of RNA and genomic DNA from one FFPE tissue sample
■ Separate eluates for DNA and RNA
■ High yields of DNA and RNA from every sample without dividing the sample or lysates
■ Efficient removal of crosslinks for each nucleic acid
A
40
40
AllPrep FFPE
RNeasy FFPE
35
30
CT value
CT value
35
25
20
15
10
A
■
■ Purification of RNA including miRNA
B
■ RNA eluates virtually free of genomic DNA due to efficient gDNA removal
AllPrep FFPE
miRNeasy FFPE
Yield, fragment length, and performance in downstream assays such as real-time PCR/RT-PCR, Pyrosequencing,
or PCR array analysis of DNA and RNA are similar to the results obtained using single nucleic acid purification
methods.
30
25
20
15
Liver Spleen Kidney Liver Spleen Kidney Liver Spleen Kidney
c-jun – RT
madh7 + RT
c-jun + RT
10
Liver
Kidney
Heart
Lung
Spleen
Total RNA was purified from various rat FFPE tissue samples using the AllPrep DNA/RNA FFPE Kit or RNeasy FFPE Kit. Real-time RT-PCR assays for madh7 and cjun each
from 30 ng RNA were performed with (+RT) or without (–RT) reverse transcriptase. The –RT samples show that RNA purified using the AllPrep DNA/RNA FFPE Kit is virtually
free of gDNA.
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual.
QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical
Services or your local distributor.
B Total RNA (including miRNA) was purified from 1 (liver, kidney, spleen) or 2 x10 µm sections (heart, lung) of various rat FFPE tissue samples using the AllPrep DNA/RNA
■
1069807
10/2011
FFPE Kit or miRNeasy FFPE Kit. The same amount of purified RNA from each sample was used as template in quantitative, real-time RT-PCR assay for miRNA miR-16.
Trademarks: QIAGEN®, QIAamp®, AllPrep®, Pyrosequencing®, QuantiTect®, RNeasy® (QIAGEN Group). Registered names, trademarks, etc. used in this document, even when
not specifically marked as such, are not to be considered unprotected by law. © 2011 QIAGEN, all rights reserved.
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