The document summarizes the AllPrep DNA/RNA FFPE Kit, which simultaneously isolates genomic DNA and total RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. FFPE samples are lysed and centrifuged to separate the RNA-containing supernatant from the DNA-containing pellet. The supernatant and pellet are then processed separately to purify high-quality RNA and DNA suitable for downstream applications like real-time PCR and pyrosequencing. Results show the kit yields RNA and DNA of sufficient quality and quantity from old FFPE samples for gene expression analysis and detection of genetic mutations.

1. Stefanie Schröer, Christiane Bäumer, Vera Holländer
QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany
110829709/2017
Simultaneous Isolation of RNA and DNA from
One FFPE Sample
Sample to Insight
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.
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.
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.
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.
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.
Genomic DNA: Yield and Fragment Length
RNA: Yield and Integrity
RNA: RT2™
FFPE PreAmp technology
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.
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. ∆∆CT
analysis shows the x-fold difference in
gene expression of the tumor sample compared with
the non-tumor sample.
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.
Genomic DNA: PCR Analysis and Pyrosequencing®
Conclusions
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
• Purification of RNA including miRNA
• RNA eluates virtually free of genomic DNA due to efficient gDNA removal
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.
Trademarks: QIAGEN®
, Sample to Insight®
, AllPrep®
, Pyrosequencing®
, QuantiTect®
, RNeasy®
, RT2™
, RT2 Profiler™
, therascreen®
(QIAGEN Group); Agilent®
(Agilent Technologies, Inc.); SYBR®
(Thermo Fisher Scientific, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked
as such, are not to be considered unprotected by law.
© 2017 QIAGEN, all rights reserved. PROM-11387-001
RNA: Real-Time RT-PCR
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.
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.
RNA yield from one (spleen, liver) or two 10 µm
sections (heart, lung) per sample determined
by OD measurement.
The same volume of RNA purified from
one 10 µm section of rat kidney was analyzed
on an Agilent®
2100 Bioanalyzer.
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.
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.
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
Incubate
supernatant
at 80°C
Lyse pellet with
proteinase K
digestion, then
incubate at 90°C
Bind genomic
DNA
Elute
Bind total RNA
Treat with
DNase, then
wash
Wash
Elute
Eluted DNAEluted RNA
Total RNA
Genomic DNA
AllPrep DNA/RNA FFPE Procedure
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 or, as a control, the QIAamp®
FFPE Tissue Kit including RNAse digestion during sample preparation. DNA yields from 20 µm sections
were determined by OD measurement. 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.
Spleen
A A AC C C M
Heart Lung
HUS1
60
50
40
30
20
10
0
x-fold difference in
gene expression
BAX
ABL1
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
KNTC1
MAD2L1
MCM3
MCM4
MCM5
MKI67
MNAT1
RAD1
RAD17
nt AllPrep RNeasy
14
12
10
8
6
4
2
0
RNA (µg)
Heart
(4 months)
Lung
(4 months)
Liver
(4 months)
Spleen
(29 months)
AllPrep FFPE RNeasy FFPE
35
30
25
20
15
10
CT value
Kidney
(3 days)
Heart
(3 days)
Liver
(19 months)
Lung
(23 months)
AllPrep FFPE RNeasy FFPE
KRAS Codon 12/13
WT: GGTGGC
KRAS Codon 61
WT: CAA, reverse
sequenced as TTG
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.
Total RNA (including miRNA) was purified from one (liver, kidney, spleen) or two 10 µm sections (heart, lung) of various rat FFPE tissue samples using
the AllPrep DNA/RNA 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.
40
35
30
25
20
15
10
CT value
Liver Spleen Kidney
madh7 + RT
Liver Spleen Kidney
c-jun + RT
Liver Spleen Kidney
c-jun – RT
AllPrep FFPE RNeasy FFPE
40
35
30
25
20
15
10
CT value AllPrep FFPE RNeasy FFPE
Liver Kidney Heart Lung Spleen
20
18
16
14
12
10
8
6
4
2
0
DNA (µg)
Lung
(6 months)
Heart
(13 months)
Spleen
(19 months)
AllPrep FFPE QIAamp FFPE