New Tools Meeting the Challenge of Your FFPE Tissue Analysis

3

Formalin-fixed, paraffin-embedded tissue (FFPET) samples are frequently used for retrospective studies, for example in oncology. They represent an increasingly important resource for medical research. Yet quantity and quality of isolated nucleic acids may confine potential downstream applications.

Achieve excellent nucleic acid purification from FFPET and pre-amplification of cDNAs from minute amounts of starting material:

� High Pure FFPET DNA Isolation Kit – For isolation of genomic DNA from formalin-fixed, paraffin-embedded tissue

High Pure FFPET RNA Isolation Kit – �

For isolation of total RNA from formalin-fixed, paraffin-embedded tissue, macrodissected and laser-microdissected samples, and biopsies

RealTime ready cDNA Pre-Amp Master & Primer �

Pools – For pre-amplification of very small quantities of cDNA for subsequent qPCR

Streamline your workflow by easily isolating and amplifying nucleic acids from your important FFPE research samples when analyzing disease progression, drug responsiveness, and toxicology.

For life science research only. Not for use in diagnostic procedures.

High Pure FFPET DNA Isolation Kit High Pure FFPET RNA Isolation Kit

RealTime ready cDNA Pre-Amp Master & Primer Pools Reveal the Archived Information in Your FFPET Samples

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Choose the High Pure FFPET DNA Isolation Kit to isolate DNA from FFPE samples:

Spin column-based purification using the High Pure FFPET DNA Isolation Kit allows you to skip DNA precipitation and organic solvent extraction, making it ideal for rapid nucleic acid isolation.

Figure 2. Isolate high-quality DNA for a variety of down-stream applications. A multiplex PCR was performed which produces PCR products of 100, 200, 300, and 400 bp amplicon length using DNA isolated from FFPET HT29 xenograft.Lanes 1 and 2: Deparaffinization using xylene. Lanes 3 and 4: Deparaffinization using hexadecan. Lanes 5 and 6: Result of multiplex PCR using an intact control

human genomic DNA isolated from whole blood.

Results: All four bands are clearly detected. This indicates high quality isolated DNA from FFPET using the High Pure FFPET DNA Isolation Kit that is suitable for demanding applications, such as array CGH and next generation sequencing.

Figure 3. Generate high-quality template DNA for excellent qPCR sensitivity. Fragment yield, purity, and size distribution are key to DNA quality. DNA must also be in sufficient quantity when amplifying a given target region during qPCR. To test this, 5 µl DNA eluate were analyzed with primers for CycA (442 bp) or myostatin (89 bp) using the LightCycler® 480 Real-Time PCR Instrument (n=4).

Results: The High Pure FFPET DNA Isolation Kit produced higher sensitivity based on the earlier quantification cycles (Cq) compared to DNA isolated using kits from all other suppliers.

400 bp

300 bp

200 bp

100 bp

MWM VIII 1 2 3 4 5 6

Figure 1. Obtain high DNA yields from FFPET samples. Histogram shows mean DNA yield in 5 µm FFPE tissue sections from three different xenografts from breast and colorectal cancer tissue research samples. As expected, a certain variation is observed between individual xenografts and individual tissue sections (n = 8).

High Pure FFPET DNA Isolation Kit

µg

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0Xenograft A Xenograft B Xenograft C

Cq

30

28

26

24

22

20Roche Supplier Q Supplier MN Supplier A

CycA Myostatin

Isolate DNA in just 3 hours, including �

deparaffinization.

Obtain high DNA yield and quality.�

Produce powerful signals downstream in qPCR, �

microarray analysis, and sequencing.

5

Table 1. Rely on DNA quality isolated with High Pure FFPET DNA Isolation Kit for whole genome amplification (WGA). 10 ng DNA were subjected to WGA using GenomePlex Whole Genome Amplification Kit from Sigma.

Results: Amplification factor of DNA isolated with High Pure FFPET DNA Isolation Kit was 1.5 times greater than of DNA isolated using the kit from supplier Q, indicating higher quality of the isolated DNA.

Table 2. Apply DNA from your FFPET samples to HRM and next generation sequencing (NGS). Total DNA was isolated from 6 individual cancer research samples using High Pure FFPET DNA Isolation Kit (BR = breast cancer, NSCLC = non-small cell lung cancer, KC = kidney cancer). Results: Applying the isolated DNA to HRM revealed two potential mutations in the TP53 gene. The HRM findings were verified and further specified by NGS using the GS FLX Instrument.

Data kindly provided by R. Loewe, GeneWake GmbH, Germany.

Sample Mutations (HRM) Mutations (NGS)

BC-1 - -

BC-2 - -

NSCLC-1 TP53 Exon 5Single base deletion

(del ch17:7578434 (T/-))

NSCLC-2 TP53 Exon 7Two SNPs (rs12947788

and rs12951053)

KC-1 - -

KC-2 - -

DNA input Amplification factor (n = 3)

Roche 10 ng 245

Supplier Q 10 ng 162

Figure 4. Apply DNA to cutting-edge applications such as methylation-sensitive high resolution melting (MS-HRM). DNA isolated from different tumor research samples was bisulfite-treated and subjected to MS-HRM on the LightCycler® 480 Instrument for the APC gene. APC is differentially methylated in tumors. Results: DNA isolated with the High Pure FFPET DNA Isolation Kit is perfectly suited to assess methylation status using MS-HRM. In this example three different methlyation levels are shown: Sample A has a methylation level of 10–100 %, sample B has a level between 0 % and 1 %, while in sample C the APC gene is not methylated.

Data kindly provided by Dr. T.K. Wojdacz, Aarhus University, Denmark.

Rel

ativ

e S

ign

al (

%)

Temperature (°C)

Normalized Melting Curves

75 75.5 76 76.5 77 77.5 78 78.5 79 79.5 80 80.5 81 81.5 82 82.5 83

100

90

80

70

60

50

40

30

20

10

0

Standards:100% methylation10% methylation1% methylation0% methylation

Samples:Samples ASamples BSamples C

Ordering Information

Product Catalog Number Pack Size

High Pure FFPET DNA Isolation Kit

06 650 767 001 Up to 50 isolations

6

High Pure FFPET RNA Isolation Kit

Experience the following benefits of the High Pure FFPET RNA Isolation Kit:

To eliminate residual DNA, an efficient on-column DNase I digestion is an integral part of the High Pure FFPET RNA Isolation Kit's protocol.

Figure 6. Obtain high RNA yields from recent and older archival FFPET samples. A) Histogram shows mean RNA yield in 10 µm FFPE colorectal cancer and normal adjacent tissue sections after the different storage times shown. Tissue sections were sequentially split between kits to avoid any bias coming from different tumor areas (n = 24).

Results: RNA isolation using supplier Q's kit resulted in lower yield.

B) RNA yields for each of the 24 replicates in the left two histogram bars* in Figure 6A. As expected, RNA yields vary between sections.

Results: The High Pure FFPET RNA Isolation Kit consistently generated higher yields compared to supplier Q's kit.

Data kindly provided by Prof. B. Molnar, Semmelweis University, Budapest, Hungary.

Yie

ld (

µg

)Yield of FFPE normal tissue (>5 years old)

12

10

8

6

4

2

0

N1_1

N5_1

N3_1

N7_1

N10_1

N11_1

N12_1

N10_2

N11_2

N12_2

N2_1

N6_1

N9_1

N4_1

N8_1

N1_2

N5_2

N3_2

N7_2

N2_2

N6_2

N9_2

N4_2

N8_2

Roche Supplier Q

B

Replicates

Figure 5. Isolate a wide range of RNA fragment sizes from 100 to 4000 bases in length. RNA was isolated from a 5 µm section of an FFPE breast tumor (KPL4 xenograft) research sample using either the High Pure FFPET RNA Isolation Kit or kits from two other suppliers. The green line shows that supplier MN’s kit purifies predominantly degraded RNA, and not longer fragment sizes. The gray graph shows that supplier Q’s kit purifies a broad spectrum of RNA fragments with a bias for short fragment sizes.

Results: In contrast, the High Pure FFPET RNA Isolation Kit (blue) purifies the entire range of RNA present, with an even distribution of fragment sizes.

[FU

]

5

4

3

2

1

0

20 30 40 50 60

Roche Supplier QSupplier MN

[s]

Yie

ld (

µg

)

12

10

8

6

4

2

0Normal adjacent

tissue

FFPET samples 5 years FFPET samples 6 months

Tumor tissue Normal adjacent tissue

Tumor tissue

A

Roche Supplier Q

Purity total RNA from formalin-fixed, paraffin- �

embedded tissue, macrodissected and laser-microdissected samples, and biopsies

Isolate RNA in just 2.5 hours, including �

deparaffinization.

Obtain high RNA yield and quality.�

Produce powerful signals downstream in �

RT-qPCR and gene expression arrays.

7

Ordering Information

Product Catalog Number Pack Size

High Pure FFPET RNA Isolation Kit

06 650 775 001 Up to 50 isolations

Table 3. Rely on high RT-qPCR sensitivity using RNA from FFPET samples with a wide range of ages. Mean absolute quantification cycle (Cq) difference between High Pure FFPET RNA Isolation Kit and supplier Q's kit. Both archive (5 years) and recent (6 months), as well as tumor (blue) and normal (black) FFPET samples, were tested. Identical amounts of RNA were subjected to RT-qPCR.

Results: Values indicate how much earlier Cqs were obtained when using the High Pure FFPET RNA Isolation Kit, indicating higher sensitivity and amplificability relative to the kit from supplier Q. Note that transcript abundance is inversely related to the Cq values obtained. Data kindly provided by Prof. B. Molnar, Semmelweis University, Budapest, Hungary.

Sample type

Archive FFPET Recent FFPET

Cq difference (n = 13 – 24)

Cq difference (n = 19 – 24)

Marker Mean Cq values using the Roche Kit were consistently earlier by:

CHI3L1 0.86 Cq

1.24 Cq

1.87 Cq

1.62 Cq

COL12A1 0.49 Cq

1.26 Cq

2.50 Cq

2.14 Cq

CXCL2 0.99 Cq

1.21 Cq

2.73 Cq

2.28 Cq

RN18S1 1.36 Cq

1.16 Cq

2.02 Cq

1.33 Cq

Figure 7. Generate high-quality template RNA with excellent sensitivity in RT-qPCR. Fragment yield, purity, and size distribution are not the only criteria of RNA quality. Isolated RNA must also be in sufficient quantity and quality when amplifying desired given target region during RT-qPCR. To evaluate the quality of the isolated RNA, a two-step RT-qPCR assay was performed. Sections from three different xenografts (HT29, MDA-MB, and KPL4) were used. 150 ng of total RNA were subjected to reverse transcription using Roche Transcriptor Universal cDNA Master, 10 ng of this preparation were applied to qPCR. An ALAS1-specific RealTime ready qPCR was performed using the LightCycler® 480 Real-Time PCR Instrument.

Results: The High Pure FFPET RNA Isolation Kit produced higher sensitivity based on the earlier quantification cycles (Cq) compared to RNA isolated using the kit from supplier Q (n = 8).

Cq

38

37

36

35

34

33

32

31

30

29

28HT29 MDA-MB KPL4

Roche Supplier Q

8

Cq Replicate 1

Cq

Rep

licat

e 2

5 10 15 20 25 30 35 40

40

35

30

25

20

15

10

5

RealTime ready cDNA Pre-Amp Master & Primer Pools

RealTime ready cDNA Pre-Amp Master

is a ready-to-use hot start reaction mix for pre-amplification of small quantities of cDNA from FFPET RNA isolations, microdissections, fine needle biopses, or rare cells for qPCR.

RealTime ready Pre-Amp Primer Pools

are the natural choice for pre-amplification, cor-responding to the primers that you use for regular qPCR assays downstream. All RealTime ready assays are customizable and function tested with detailed assay descriptions and bioinformatic background information available.

The entire workflow for gene expression analysis in FFPET samples can be performed using proven, optimized technologies provided by Roche Applied Science.

Figure 8. Achieve highly reproducible qPCR results after pre-amplification. Total RNA was isolated from FFPET using the High Pure FFPET RNA Isolation Kit and reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit. Two pre-amplifi-cation reactions for 19 different targets from 5 ng of one cDNA pool were performed. Each data point reflects one target.

Results: Cq values are highly reproducible over a broad gene expression range.

RealTime ready Pre- Amp Primer Pool or

Single Assays

RealTime readycDNA Pre-Amp

Master

Diluted pre-ampcDNA

LightCycler® 480Probes Master

qPCR of RealTime ready CustomPanel with pre-amplified cDNA

Amplification PCRfor 11 – 14 cycles

cDNA Aliquot

cDNA Synthesis

RNA Isolation

RealTime readyCustom Panel

e.g., using High PureFFPET RNA Isolation Kit

e.g., using Transcriptor FirstStrand cDNA Synthesis Kit

e.g., using LightCycler®

480 Instrument

Overcome limiting amounts of cDNA (down to �

1 ng) when analyzing a large number of tar-gets.

Achieve an approx. 4000-fold amplification of �

your targets of interest in less than 1 hour.

Enjoy uniform, highly reproducible pre-amplifi-�

cation without the bias that can be observed using whole transcriptome amplification (WTA) technologies.

R2 = 0.993

9

Cq with pre-amplification

Cq

wit

ho

ut

pre

-am

plif

icat

ion

5 10 15 20 25 30 35 40

40

35

30

25

20

15

10

Table 4. Comparison of RealTime ready cDNA Pre-Amp Master to similar kits from other suppliers. For each kit, 48 different genes were tested in four- to six-fold replicates. Standard deviation (SD) and ∆Cq between results (pre-amplified versus not pre-amplified) were calculated for all genes and replicates tested.Results: The RealTime ready cDNA Pre-Amp Master exhibited lowest SD as well as highest ∆Cq for RT-qPCR of all the kits tested.

Data kindly provided by Prof. M. Kubista, Göteborg, Sweden.

Figure 11. Robust pre-amplification is even possible with cDNA from induced pluripotent stem (iPS) cells. Total RNA was isolated using RealTime ready Cell Lysis Kit and reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit. cDNA (pre-amplified vs. not pre-amplified) was subjected to qPCR on the LightCycler® 480 System. Pre-amplified samples were 1:40 diluted before qPCR. Each data point reflects one of the 29 target assays.

Results: Despite dilution, values of the pre-amplified targets shift approx. 5 Cqs earlier. This shift is highly reproducible for all tar-gets analyzed.

Figure 9. Trust in unbiased preamplifi cation of your qPCR targets. Total RNA was isolated from FFPET using the High Pure FFPET RNA Isolation Kit and reverse transcribed using the Tran-scriptor First Strand cDNA Synthesis Kit. 5 ng of cDNA (pre-ampli-fi ed vs. not pre-amplified) were subjected to qPCR on the Light-Cycler® 480 System. Pre-amplifi ed samples were 1:40 diluted before qPCR. Each data point refl ects one target.

Results: Despite dilution, values of pre-amplifi ed targets shift approx. 5 Cqs earlier. This substantial shift is highly reproducible for all targets analyzed.

Figure 10. Enjoy a broad range of possible cDNA sample inputs. 1 ng (x-axis) and 250 ng (y-axis) of cDNA were subjected to pre-amplifi cation and subsequent qPCR using the LightCycler® 480 System. The 96 individual assays of the RealTime ready Human Apoptosis Panel were used. Pre-amplifi ed samples were 1:40 dilut-ed before qPCR. Each data point refl ects one target assay.

Results: RT-qPCR results are highly reproducible over a broad range of both cDNA starting material and gene expression levels.

Mean SD Mean ∆Cq

Without pre-amplification 0.40 -

RealTime readycDNA Pre-Amp Master

0.18 -6.99

Supplier L 0.22 -6.43

Supplier Q 0.27 -6.51

Ordering Information

Product Catalog Number Pack Size

RealTime ready cDNA Pre-Amp Master 06 720 455 001 For 40 cDNA multiplex pre-amplifications

RealTime ready Pre-Amp Primer Pool 06 725 309 001 For 40 cDNA multiplex pre-amplifications

Cq 25

0 ng

cD

NA

Cq 1 ng cDNA

5 10 15 20 25 30 35 40

30

25

20

15

10

5

0

R2 = 0.9866

Cq

wit

ho

ut

pre

-am

plif

icat

ion

Cq with pre-amplification15 20 25 30 35

40

35

30

25

20

R2 = 0.9629R2 = 0.9923

10

Product Catalog Number Pack Size

LightCycler® 480 Instrument II 05 015 278 001 1 instrument, 96-well version

LightCycler® 480 Probes Master 04 887 301 001 For up to 10 500 reactions, 20 µl final volume

LightCycler® 480 High Resolution Melting Master 04 909 631 001 For up to 500 reactions, 20 µl final volume

LightCycler® 480 Multiwell Plate 96, white 04 729 692 001 5 10 plates with sealing foils

RealTime ready Cell Lysis Kit 05 943 523 001 500 lysis reactions with a final reaction volume of 40 µl each

RealTime ready Human Apoptosis Panel, 96 05 392 063 001 2 plates, each containing 96 assays

Trancriptor First Strand cDNA Synthesis Kit 04 896 866 001 1 kit for up to 100 reactions

GS FLX+ Instrument 06 372 279 001 1 instrument plus accessories

HIGH PURE , LIGHTCYCLER, GS FLX and REALTIME READY are trademarks of Roche.NOTICE: This product may be subject to certain use restrictions. Before using this product please refer to the Online Tech-nical Support page (http://technical-support.roche.com) and search under the product number or the product name, whether this product is subject to a license disclaimer containing use restrictions.

For life science research only. Not for use in diagnostic procedures.

Additional Products

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