Kit for RNA Extraction from Paraffin-Embedded Tissue Sections

ISOGEN PB Kit
Manual (Second edition)
Code No. 315-06421

NIPPON GENE CO., LTD.

Table of contents
I

Product description

1

IIBefore using

1

IIIContents of kit

2

IVStorage

2

V

Precautions

2

VI

Protocol3

VII Examples of experiments

8

ISOGEN PB Kit (ver.2)
111006KH

I

Product description

The ISOGEN PB Kit is for extracting RNA from paraffin-embedded tissue sections.
RNA can be extracted in a short time (about 2 hours) by the following simple manipulations:
Deparaffinization → Proteinase K treatment → RNA extraction (ISOGEN-LS).
■ Use of extracted RNA・ Detection of RNA virus by RT-PCR.
・ Detection of mRNA by RT-PCR.
・ Checking the preservation of RNA in paraffin-embedded tissue sections, etc.

IIBefore using

Since RNA extracted from paraffin-embedded tissue sections is progressively degraded in
the process before the embedding, the quality of the RNA is less than that extracted fromfresh tissue.
The total RNA extracted from the fresh tissue of a mammal demonstrates clear bands of 28SrRNA and 18S rRNA in electrophoresis, and the ratio is about 2: 1.

However, when the RNA

is extracted from paraffin-embedded tissue, the result is not necessarily the same.

Actually,in an extraction experiment using paraffin-embedded tissue of a mouse, almost no 28S rRNA
was found. Therefore, it is suggested that the RNA is degraded in the process before theembedding.

Further, the extent of RNA degradation is greatly dependent on the condition of

the paraffin-embedded block.

The presence of 18S rRNA is clearly confirmed in someblocks but in other blocks the degradation is progressed so much that the presence of 18SrRNA cannot be confirmed.
Therefore, we consider the persistence of 18S rRNA as one of the criteria of judgment. We
recommend this as a guide, if the preservation of RNA is important in cases such as In situ
hybridization.
Further, even if RNA is degraded (even if 18S rRNA is not confirmed), often the gene can bedetected by RT-PCR, and normally the completeness of RNA is not required.

However,since the amplification efficiency of the PCR from paraffin-embedded tissue sections is lower
compared to normal PCR, make sure to set the primers so that the amplified size is not too
big (not more than 400 bp, and preferably not more than 200 bp).

-1-ISOGEN PB Kit (ver.2)
111006KH

III

Contents of kit( For 20 extractions )Proteinase K (20 mg/ml)

100 µl × 1

Extraction Buffer *3 ml × 1

ISOGEN-LS

10 ml × 1

Ethachinmate

60 µl × 1

Deoxyribonuclease (RT Grade)

20 µl × 1

10×DNase (RT Grade) BufferⅡ

100 µl × 1

Stop Solution (RT Grade)

100 µl × 1

DEPC treated Water

500 µl × 2* White crystals may appear in Extraction Buffer but this is not a quality problem.
In such cases, incubate at 37-50°C to completely dissolve the crystals and then use.

IVStorage

Store at －20°C
・ Extraction Buffer can be stored at room temperature.
・ ISOGEN-LS can be stored at 2-10°C.

VPrecautions• This product is a reagent for research and cannot be used for medical or other objectives.
Also, this product should be handled only by persons having a basic knowledge about
reagents.
• ISOGEN-LS is a deleterious substance not for medical use (phenol formulation), andshould be handled with great care.
• When using, wear appropriate protective gear (gloves, goggles, etc.).
• Ventilate the area well so as not to inhale the vapor.
• If the product enters eyes or adheres to skin, wash with large amounts of water for atleast 15 min and consult a doctor.
• Handle this product in accordance with the descriptions in the manual.
• We are not responsible for problems caused if this product is not handled in accordancewith the manual.
• The product safety data sheet (SDS) can be reviewed on our website.
URL https://www.nippongene.com/-2-ISOGEN PB Kit (ver.2)
111006KH

VIProtocol< Reagents necessary in addition to the kit >□ Lemosol or Lemosol A (Xylene may be used)□ Ethanol□ Chloroform□ Isopropanol< RNA extraction protocol >Paraffin-embedded tissue sections (＜25 mm2 × 10 µm × 4 sections) *1) in 1.5 ml tube *2)
Lemosol or Lemosol A *3) 800 µl
Mix by inversion (about 10 times).
Allow to stand at room temperature for 1 min.
(May be omitted) *4)

100% Ethanol 400 µl
Mix by inversion.

Centrifugation (12 K×g, 2 min, room temperature)
Precipitates *5)
100% Ethanol 1 ml
Mix by inversion (about 10 times)
Allow to stand at room temperature for 1 min.
Centrifugation (12 K×g, 2 min, room temperature)
Precipitates *5)
Dry *6)Extraction Buffer 150 µl
Proteinase K (20 mg/ml) 5 µl
Pipetting or light vortex *7)
Incubate at 50°C for 15 min (～16 hrs) *8)(Continue to the next page)-3-

ISOGEN PB Kit (ver.2)
111006KH

(continued from the previous page)
ISOGEN-LS 450 µl
vortex 5 sec (～20 sec) *9)
Allow to stand at room temperature for 5 min.
Chloroform 120 µlvortex 15 sec
Allow to stand at room temperature for 3 min.
Centrifugation (12 K×g, 15 min, 4°C)
Aqueous phase (350 μl)
Ethachinmate *10) 3 µl
Isopropanol 300 µl
vortex 5 sec
Allow to stand at room temperature for 5 min (～10 min).
Centrifugation (12 K×g, 10 min, 4°C)
Precipitates
70% Ethanol 1 ml
Mix by light inversion
Centrifugation (12 K×g, 5 min, 4°C)
Precipitates
Dry *11)
DEPC treated Water ～20 µl
RNA solution-4-ISOGEN PB Kit (ver.2)
111006KH

*1) About 4 pieces of paraffin-embedded tissue sections (＜25 mm2 × 10 µm) are used perextraction.

Remove excess paraffin around the tissue using a utility knife or the like, thenslice the tissue.

Since paraffin is soluble in Lemosol, Lemosol A and Xylene, it is notnecessary to completely remove paraffin.
Also, when RNA is extracted from a tissue section on a slide glass, scrape the tissue from theslide glass using a microspatula or the like before deparaffinization.
*2) We recommend use of 1.5 ml volume tubes made of clear polypropylene.

Before use

confirm that the tubes can withstand centrifugal force (12 K×g) and ISOGEN-LS (Phenol).
*3) Xylene may be used but we recommend use of Lemosol or Lemosol A because forimproved safety.
*4) At this step, ethanol addition may be omitted. When the sample tends to float in thesupernatant, the ethanol addition allows tissue to more easily precipitate and makes
manipulation easier.
However, when a sample containing a large amount of paraffin is used, the ethanol addition
may cause deposition of paraffin (white turbidity). Therefore, we recommend centrifuging
without adding ethanol. If paraffin deposition is observed, centrifuge the sample to remove
the supernatant, and then add Lonosol or Lemosol A again and perform the manipulation
thereafter without adding “100% Ethanol 400 µl”.
*5) Remove the supernatant with a micropipette. Since the tissue may float at this step, be
careful not to suck in.
*6) Air dry or vacuum dry until Ethanol is not visible.
*7) Make sure that the solution is foamy.
*8) RNA extraction efficiency may be improved by prolonging the reaction time ofProteinase K when a sample having a large tissue size is used, or when RNA yield is low.
*9) Mix until Extraction Buffer and ISOGEN-LS become homogeneous.
*10) Ethachinmate is an acrylamide polymer carrier solution used for precipitating nucleic
acid with ethanol.

RNA can be efficiently recovered by adding Ethachinmate, and theprecipitation can be visually confirmed.
*11) Air dry ethanol until Ethanol is no longer visible.

However, when RNA precipitates arecompletely dried, RNA may become difficult to dissolve.
-5-ISOGEN PB Kit (ver.2)
111006KH

< Handling of extracted RNA >・ When electrophoresis and concentration measurement are performed, use the preparation
as-is.
・ When the RNA prepared by this product is used for RT-PCR, treat the sample with
Deoxyribonuclease (RT Grade) attached with the kit.
< Preparation of template RNA solution for RT-PCR:
Extracted RNA solution

DNase treatment >(0.1 - 5 µg)

10×DNase (RT Grade) BufferⅡ

2.5 µl

Deoxyribonuclease (RT Grade)

0.5 µlDEPC treated Water
25 µl *1)

Total
↓Enzyme reaction 37°C 15 min *2)↓ *3)↓Stop Solution (RT Grade)

2.5 µl↓Heat treatment

70°C 10 min *4)↓RT-PCR *5)*1) When the reaction is carried out in a system other than 25 µl, the ratio ofDeoxyribonuclease (RT Grade): 10×DNase (RT Grade) BufferⅡ: Stop Solution (RT Grade)should always be the volume ratio of 1: 5: 5. The effect of Stop Solution (RT Grade) may not
be sufficient in other ratios. It is very important to chelate Ca2+ in the reaction solution for
stopping DNase reaction.
*2) Do not react for 15 min or longer.
*3) When stopping at this time, DNase should be inactivated and removed by aphenol/chloroform treatment. In such cases, manipulations after adding Stop Solution (RTGrade) are unnecessary (see next page).
*4) RNA is unstable at a high temperature.

Perform the heat treatment at 70°C.

At thistemperature DNase is not completely inactivated but it is not a problem in the reverse
transcription reaction.
*5) After the reverse transcription reaction, stop the reaction of the reverse transcription
enzyme by a heat treatment at 95°C for 5 min.
-6-ISOGEN PB Kit (ver.2)
111006KH< Precautions for DNase treatment >・ Remove DNA by the reaction described above as quickly as possible before RT-PCR.
・ When RNA treated with DNase needs to be stored for a long period of time, perform theinactivation and removal of DNase by the phenol/chloroform treatment.
・ When electrophoresis is performed for RNA treated with DNase, perform thephenol/chloroform treatment and recovery by ethanol precipitation of the RNA.

Themobility is affected abnormally by the effect of 10×DNase (RT Grade) BufferⅡand StopSolution (RT Grade).
・ When DNase is inactivated and removed by the phenol/chloroform treatment, the additionof Stop Solution (RT Grade) and the heat treatment at 70°C for 10 min are unnecessary.
( Example of DNase inactivation by phenol/chloroform treatment )Add 75 µl of DEPC treated water to 25 µl of DNase treated RNA solution to bring thevolume to 100 µl, and then add an equal volume (100 µl) of phenol/chloroform *1) andmix.
↓Centrifuge (12 K×g, 5-10 min, room temperature) and transfer the upper layer(aqueous phase) to a new tube.
↓
Add an equal volume (100 µl) of chloroform to the upper layer and mix.
↓Centrifuge (12 K×g, 5-10 min, room temperature) and transfer the upper layer(aqueous phase) to a new tube.
↓Add 1/10 volume (10 µl) of 3 M sodium acetate solution and 2.5 fold volume (250 µl)of ethanol to the upper layer, mix and allow to stand at room temperature for 5-10min (or at -20°C for 30 min).
↓Centrifuge (12 K×g, 10 min, 4°C) and remove the supernatant.
↓
Wash the precipitates with 70% ethanol (add about 1 ml, mix lightly by inversion,
centrifuge and remove the supernatant).
↓Air dry the precipitates for 5-10 min and dissolve in DEPC treated Water.
↓RNA solution*1) "Phenol/Chloroform/Isoamyl alcohol (25: 24: 1)" (Code No. 311-90151)-7-

ISOGEN PB Kit (ver.2)
111006KH

VII

Examples of experiments< RNA extraction from paraffin-embedded mouse submaxillary gland sections >RNA is extracted from 10 µm × 4 sections of paraffin-embedded mouse submaxillary glandaccording to the protocol and a quarter of the RNA solution (5 µl) is electrophoresed in 1%
formalin denatured Agarose S.
1

2
1. RNA isolated from fresh tissue(extracted with ISOGEN)
2. RNA extracted from paraffin-embedded tissue sections(Extracted with ISOGEN PB Kit)

< Detection of the mouse glyceraldehyde-3-phosphate dehydrogenase (Gapd) gene byRT-PCR >After treating 500 ng of the RNA extracted from paraffin-embedded mouse submaxillary glandsections with DNase according to the protocol, Exon 5 (258 bp) of the mouse Gapd gene was
detected by RT-PCR.
Reverse transcription reaction
Reaction solution composition

Reverse transcription reaction condition

10×Reaction Buffer *2 µl

Total 20 μl

25 mM MgCl2 *4 µl

2.5 mM dNTP *4 µl

25℃ 10 min

50 µM Random Nonamer *1 µl48℃ 30 min

RNase Inhibitor (20 U/µl) *0.5 µl

95℃ 5 minEuroScript RT (50 U/µl) *0.5 µlRNA treated with DNase

8 µl

Total

20 µl

1st strand cDNA

* Reverse Transcriptase qPCR Core Kit (EUROGENTEC INC.) was used for reagents ofreverse transcription reaction.

-8-ISOGEN PB Kit (ver.2)
111006KH

PCR
Reaction solution composition

PCR condition

10×Gene Taq Universal Buffer

2.5 µl

94℃ 2 min

2 µl

95℃ 15 sec

3 μM Gapd Primer Mixture

2.5 µl

60℃ 30 sec

Gene Taq NT (5 U/µl)

0.25 µl

72℃ 30 sec2.5 mM dNTP Mixture

1st strand cDNA

5 µl

Distilled Water

12.75 µl

Total
1×3525 µl
2345

1: OneSTEP Marker 5 (ΦX174 / HincⅡdigest)
2: No template
3: RNA for which no reverse transcription reaction
was performed.
4: RNA for which reverse transcription reaction
was performed.
5: OneSTEP Marker 5 (ΦX174 / HincⅡdigest)

One half volume (12.5 µl) of the PCR productwas electrophoresed in 2% Agarose S.

< RT-PCR for microscale sample >When the extracted RNA cannot be confirmed in electrophoresis or is not more than 10 ng,we recommend performing a reverse transcription reaction by adding RTmate (Code No.
315-05941).

The detection limit sensitivity of RT-PCR is increased by adding RTmate.

Detailed information about RTmate such as the sequence can be referred on our website(http://www.nippongene.com/).

ISOGEN PB Kit (ver.2)
111006KH-9-

NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel
076-451-6548
URL
https://www.nippongene.com/

・The information in the descriptions, specifications and the prices of the products may be changed without priornotification.

ISOGEN PB Kit (ver.2)
111006KH