HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

RNA EXTRACTION HUSM/HEMA-UPT/STM-M1 VERSION : VERSION DATE : 2 01.03.2011

APPROVED BY :

.. ASSOC PROF DR ROSLINE HASSAN HEAD OF HAEMATOLOGY DEPARTMENT CONTROLLED COPY NO : 4 REGISTERED HOLDER HAEMATOLOGY LABORATORY

RECORD OF REVIEW/AMMENDMENT DATE

01.03.2011 01.03.2011

VERSION NO.
2 2

DETAIL OF AMMENDMENT
Page 3: added the specimen storage condition at NB: Working with RNA, 6 : edited the limitation ratio.

BY
Selamah Ghazali Selamah Ghazali

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. RNA EXTRACTION HUSM/HEMA-UPT/STM-M1 VERSION : VERSION DATE : 2 01.03.2011

PREPARED BY DESIGNATION CHECKED BY DESIGNATION AUTHORISED BY DESIGNATION

: SELAMAH GHAZALI : TECHNICAL MANAGER / MEDICAL LAB TECHNOLOGIST U36

: : : : ANG CHENG YONG TRAINING OFFICER / SCIENTIFIC OFFICER ASSOC PROF DR ROSLINE HASSAN HAEMATOLOGIST / LAB DIRECTOR / HEAD OF DEPARTMENT

1.

OBJECTIVE
To acquire RNA from blood and bone marrow samples for molecular studies

2.

METHOD
QIAamp RNA Mini Kit by Qiagen.

3.

PRINCIPLE
QIAamp spin columns represent a technology for total RNA preparation that combines the selective binding properties of a silica-gel-based membrane with the speed and conveniene of microspin technology. A specialized high-salt buffering system allows RNAs longer than 200 bases to bind to the QIAamp membrane. During the QIAamp procedure for purification of RNA from blood, erythrocytes are selectively lysed and leukocytes are recovered by centrifugation. The leukocytes are then lysed using highly denaturing conditions that immediately inactive Rnases, allowing the isolation of intact RNA. After homogenization of the lysate by a brief centrifugation through a QIAshredder TM spin column, ethanol is added to adjust binding conditions and the sample is applied to the QIAamp spin column.RNA is bound to the silica-gel membrane during a brief centrifugation step. Contaminants are washed away and total RNA is eluted in 30ul or more of Rnase-free water for direct use in any downstream application. Since the procedure relies on intact leukocytes, frozen blood cannot be used. QIAamp RNA Blood Kits enrich for RNAs larger than 200 nucleotides since small RNAs such as 5.8s RNA,5S RNA, and tRNA (approximately 160,120, and 70-90 nucleotides in length ,respectively), which make up 15-20% of the total RNA, do not bind in quantity under the conditions used. Thus the size distribution of RNA isolated with the QIAamp cushion, where small RNAs do not sediment efficiently. Phenol-based procedures copurify RNAs <200 nt, accounting for the 20% higher yield of RNA purified using phenol-based procedures in comparison to QIAamp based procedures.

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. RNA EXTRACTION HUSM/HEMA-UPT/STM-M1 VERSION : VERSION DATE : 2 01.03.2011

NB: Working with RNA Extracted RNA from samples preferably stored and transported at room temperature, within 48 hours of collection. Whole blood or bone marrow specimen collected in EDTA stored at 4oC- 8 oC. . It is extremely important to minimize the presence and activity of RNases during RNA extraction. RNases are found on all glassware and plasticware, electrophoresis tanks, solutions, hands, dust etc and liberated during cell lysis. Gloves must ALWAYS be worn and changed frequently during RNA extraction. All glassware and solutions (where possible) must be prepared with 0.1 % DEPC, incubated overnight at room temperature and then autoclaved to inactivate the DEPC. Placticware produced commercially and handled only with gloves on can be assumed to be RNase free. ALL waste from this method, including pipette tips and tubes. MUST be disposed of into screw cap waste containers WITHIN the bench RNA extraction. These must be sealed and disposed of in the hazardous waste bin.

4.

REQUIREMENTS 4.1 EQUIPMENT

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Refrigerated Centrifuge Sterile 15 mL blue cap centrifuge tube. Sterile 1.5 mL tubes Sterile pipette tip yellow Sterile pipette tip blue Sterile 2.0 mL Collection tube Pipette volume 100- 1000 L Pipette volume 10 -100 L Water bath (37oC) Deep Freezer -70 oC Thermo / Revco. Mixer Microtube rack. Timer.

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. 4.2
4.2.1

RNA EXTRACTION HUSM/HEMA-UPT/STM-M1

VERSION : VERSION DATE :

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REAGENTS
Test specimen: EDTA anticoagulated whole blood and bone marrow NB: Samples processed within 48 hours of collection and stored/ transported at room temperature. If outside this criteria suggest recollection and report requesting RNA as No Result.

4.2.2

QIAamp RNA blood Mini Kits. 1. 2. 3. 4. 5. 6. 7. QIAamp Spin Columns (clear) QIAshredder TM Spin Columns ((lilac) Buffer EL Buffer RLT Caution: Corrosive and toxic.Please refer to MSDSs before handling. Buffer RW1 Caution: Flammable. Please refer to MSDS before handling. Buffer RPE Rnase-free water

Storage QIAamp RNA Blood Mini Kits should be stored dry, at room temperature (15-25oC) and stable for at least 9 months under these conditions.

4.2.3. Ethanol (99.7-100% v/v)- Analar BDH. 4.2.4. 70 % ethanol EtOH- Analar BDH Sterile water. Measure out 35-mL absolute ethanol in sterile 50 mL blue top centrifuge tube. Make the final volume up to exactly 50 mL with sterile water. Mix well by inversion. 4.2.5. 14.5 M -mercaptoethanol (-ME ) (commercially available solutions are
usually14.5 M )

Caution: -ME must be added to Buffer RLT before use. -ME is toxic; dispense
in a fume hood and wear appropriate protective clothing.

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. RNA EXTRACTION HUSM/HEMA-UPT/STM-M1 VERSION : VERSION DATE : 2 01.03.2011

4.3

SAMPLE COLLECTION, STORAGE, AND HANDLING.

QiaAMP RNA Blood Mini Kits are designed for isolation of total cellular RNA from fresh, whole blood and bone marrow. Whole blood and bone marrow should be collected in the presence of an anticoagulant, preferably EDTA, although other anticoagulants such as citrate, heparin, or ACD (acid citrate dextrose) can also be used. For optimal results, blood samples should be processed within a few hours of collection. mRNAs from blood cells have different stabilities. mRNAs of regulator genes have shorter half-lives than mRNAs of housekeeping genes. To ensure that the isolated RNA contains a representative distribution of mRNAs, blood samples should not be stored for long periods before isolating RNA. To prevent RNA contamination, please refer to General Guideline in appendix 4. Note : The QIAamp RNA Blood Mini Kit cannot be used for frozen blood samples.

5.
NO 5.1

PROCEDURE
ACTIVITY Important notes before starting: Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of ethanol (96-100 % ) to obtain a working solution. Buffer RLT may form a precipitate upon storage. If necessary, warm to redissolve. -Mercaptoethanol (-ME) must be added to Buffer RLT before use. Add 10 L of -ME per 1 mL of Buffer RLT. Buffer RLT is stable for 1 month at room temperature (15 -25oC) after addition of -ME. After erythrocyte lysis, all steps of this protocol should be performed at room temperature (15-25oC), as quickly as possible. Cell lysates (in Buffer RLT) can be stored at -70oC. To process frozen lysates, thaw and incubate for 10 min at 37oC to ensure all salts have dissolved. Continue with step 5.8. The use of frozen leukocyte pellets (without addition of Buffer RLT) is not recommended. Volume of whole blood use will depend on TWBC. IF TWBC less than 30.0 x 10^9/ TWBC used 2 ml of blood. Mix 1 volume of whole blood with 5 volume of buffer EL in 15 mL falcon tube. For optimal results, the volume of the mixture (blood + Buffer EL) should not exceed of the volume of the tube to allow efficient mixing. For example, add 5
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RESPONSIBILITY MLT/SO

5.2

MLT/SO

HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. RNA EXTRACTION HUSM/HEMA-UPT/STM-M1 VERSION : VERSION DATE : 2 01.03.2011

mL of Buffer EL to 1 mL of whole blood, and mix. NO 5.3 ACTIVITY Incubate for 20 min on ice. Mix by vortexing briefly 2 times during incubation. The cloudy suspension becomes translucent during incubation, indicating lysis of erythrocytes. 5.4 Centrifuge at 500 rpm for 10 min at 4oC, and completely remove and discard supernatant using sterile pipette. Leukocytes will form a pellet after centrifugation. Ensure supernatant is completely removed. Trace amounts of erythrocytes, which give the pellet a red tint, will be eliminated in the following step. 5.5 Add Buffer EL to the cell pellet (use 2 volumes of Buffer EL per volume of whole blood used in step 5.2). Resuspend cells by vortexing briefly. For example, add 2 mL of Buffer EL per 1 mL of whole blood in step 5.2. 5.6 Centrifuge at 500rpm for 10 min at 4oC, and completely remove and discard supernatant using sterile pipette tip. (if necessary repeat procedure 5.5 and 5.6 again one time ) Note : Incomplete removal of the supernatant will interfere with lysis and subsequent binding of RNA to the QIAamp spin column, resulting in lower yield. 5.7 Add Buffer RLT to pelleted leukocytes according to the table below. Vortex or pipette to mix. When not using healthy blood, refer to number of leukocytes to determine the volume of Buffer RLT required. Buffer RLT disrupts the cells. No cell clumps should be visible before you proceed to the homogenization step. Vortex or pipette further to remove any clumps.
Buffer RLT (ul) 350 600 No.of leukocytes less 30.0x10^9/L more than 30.0x10^9/L

RESPONSIBILITY MLT/SO

MLT/SO

MLT/SO

MLT/SO

MLT/SO

Note: Cell lysate (in Buffer RLT) can be stored at -70oC. To process frozen lysates, thaw and incubate for 10 min at 37oC to ensure all salt have dissolved. 5.8 5.8.1 Process lysate Manually MLT/SO

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

NO 5.8.1.1

RNA EXTRACTION HUSM/HEMA-UPT/STM-M1

ACTIVITY

VERSION : VERSION DATE :

2 01.03.2011
RESPONSIBILITY MLT/SO

Pipette lysate directly into a QIAshredder spin column in a 2-mL collection tube and centrifuge for 2 min at full speed to homogenize. Discard QIAshredder spin column and save homogenized lysate To avoid aerosol formation, adjust pipet to >/- 750 L to ensure that the lysate can be added to theQIAshredder spin column in a single step. If too much cells have been used, after homogenization the lysate will be too viscous to pipet. Add 1 volume (350 L or 600 L) of 70 % Ethanol to the homogenized lysate and mix well by pipetting. Do not centrifuge. A precipitate may form after the addition of ethanol. This will not affect the QIAamp procedure.

5.8.1.2

MLT/SO

5.8.1.3

Carefully pipette sample, including any precipitate which may have formed, into a new QIAamp spin column sitting in a 2-mL collection tube (provided) without moistening the rim. Centrifuge for 15 sec at 10,000 rpm. Discard flow-through* and collection tube. Note: Maximum loading volume is 700 L. If the volume of the sample exceeds 700 L, successively aliquots onto the QIAamp spin column and centrifuge as above.

MLT/SO

5.8.1.4

Tranfer the QIAamp spin column into a new 2-mL collection tube(provided). Apply 700 L Buffer RW1 to the QIAamp spin column and centrifuge for 15 sec at 10,000 rpm to wash. Discard flow-through* and collection tube. * Flow-through contains Buffer RW1 or RLT and is therefore incompatible with bleach

MLT/SO

5.8.1.5

Place the QIAamp spin column into a new 2-mL collection tube (provided). Pipette 500 L Buffer RPE into the QIAamp spin column and centrifuge for 15 sec at 10,000 rpm. Discard flow-through * and collection tube.

MLT/SO

5.8.1.6

Carefully open the QIAamp spin column and add 500 L Buffer RPE. Close the cap and centrifuge at full speed for 3 min. Note: Some centrifuge rotors may distort slightly upon deceleration. Resulting in flow-throught, containing Buffer RPE, contacting the QIAamp spin column. Removing the QIAamp spin column and collection tube from the rotor may also cause flow-through to come into contact with the QIAamp spin column.

MLT/SO

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

NO 5.8.1.7

RNA EXTRACTION HUSM/HEMA-UPT/STM-M1

ACTIVITY

VERSION : VERSION DATE :

2 01.03.2011
RESPONSIBILITY MLT/SO

Place the QIAamp spin column into a new 2-mL collection tube(provided). And discard the old collection tube with the filtrate.Centrifuge at full speed for 1 min. Tranfer the QIAamp spin column into a 1.5 mL microcentrifuge tube (provided) and pipette 50 L of Rnase-free water (provided) directly onto the QIAamp membrane. Centrifuge for 1 min at 10,000 rpm to elute. Process lysate by using QIAcube (automation)
Note: Before proceeding, please familiarize yourself with the features of the

5.8.1.8

MLT/SO

5.8.2

MLT/SO

5.8.2.1

instrument (Appendix 1) Place rotor adapters into the rotor adapter holder according to the number of samples and position as shown in the QIAcube loading chart (Appendix 2). Place spin column into position label no 1 and slot the spin column lid into L1 as shown in Rotor Adapter Diagram (Appendix 3). Place QIAshredder TM Spin Columns into position label no 2 and slot the QIAshredder TM spin column lid into L2 as shown in Rotor Adapter Diagram (Appendix 3). Label the 1.5 mL microcentrifuge tube according to sample and place it into position label no 3 and slot the microcentrifuge lid into L3 as shown in Rotor Adapter Diagram (Appendix 3). Open the instrument door Fill up the tip rack (light-gray colored) with 1000 L disposable filter-tips Prepare the reagent bottle rack and place i) ii) iii) iv) 70% ethanol (with bottle without cap) into position no 2 Buffer RW1 (with bottle without cap) into position 4 Buffer RPE (with bottle without cap) into position 5 RNase-free water (with bottle without cap) into position 6.

MLT/SO

5.8.2.2

MLT/SO

5.8.2.3

MLT/SO

5.8.2.4

MLT/SO

5.8.2.5 5.8.2.6 5.8.2.7

MLT/SO MLT/SO MLT/SO

5.8.2.8

Install the shaker adapter (marked with 2) in the instrument by using the Allen key. i) Place the RLT into the Shaker rack according to the number of samples and position as shown in the QIAcube loading chart (Appendix 2). Place tube lid into the slot at the edge of the shaker rack.

MLT/SO

ii)

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

iii) 5.8.2.9 NO 5.8.2.10 5.8.2.11 5.8.2.12 5.8.2.13 5.8.2.14 5.8.2.15 5.8.2.16 Close the instrument door. Switch on the instrument at the power switch. Select the RNA from the main menu. Select QIAamp RNA Blood Mini Select 350 L lysed leukocytes Select STANDARD Press START Note: If an error is message displayed in the touchscreen. Resolve the error so that the protocol run can continue. When the protocol run has finished, a message is displayed in the touchscreen confirming that the samples have been processed. Follow the instructions in the touchscreen for worktable cleanup. 5.8.2.17 5.9 5.10 . 6. Switch off the power switch RNA concentration is quantitation by spectrophotometry. (refer STM Nucleic Acid Quantitation by spectrophotometry) Total RNA may be stored at 20oC or 70oC in water. Under these conditions at QIAGEN, no degradation of RNA has been detected even after 1 year. MLT/SO MLT/SO MLT/SO

RNA EXTRACTION HUSM/HEMA-UPT/STM-M1

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Transfer the shaker rack into the shaker adapter. MLT/SO RESPONSIBILITY MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO MLT/SO

Transfer the rotor adapters from 5.8.2.1 into centrifuge ACTIVITY

LIMITATION
Pure RNA has an A260/ A280 ratio , ranging from 1.9-2.1.

7.

RESULT / NTERPRETATION Concentration of RNA:.ng/ul.

8.

REFEERENCES
1. QIAamp RNA Blood Mini Handbook (Second Edition, September 2006)
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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. RNA EXTRACTION HUSM/HEMA-UPT/STM-M1 VERSION : VERSION DATE : 2 01.03.2011

Appendix 1: Features of the instrument (QIAcube)

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

Appendix 2: Loading Chart

RNA EXTRACTION HUSM/HEMA-UPT/STM-M1

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2 01.03.2011

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO.

Appendix 3: Rotor Adapter

RNA EXTRACTION HUSM/HEMA-UPT/STM-M1

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2 01.03.2011

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. RNA EXTRACTION HUSM/HEMA-UPT/STM-M1 VERSION : VERSION DATE : 2 01.03.2011

Appendix 4: GENERAL GUIDELINES: PREVENTING CONTAMINATION IN THE PCR LABORATORY

1. Definition:

Contamination: accidental contact or mixing of the sample with exogenous material (nucleic acids or microbial organisms) that make the sample impure or corrupt. 2. Laboratory practices designed to exclude cross-contamination DNA/RNA in routine PCR laboratory 2.1 Cultivating laboratory habits that prevents cross-contamination Preventing cross-contamination requires the cooperation of all laboratory personnel. Only if all the people in the laboratory follow good work habit, will the laboratory be free of contamination. Example: Never reverse the direction of the workflow (e.g., by transporting amplified material into the DNA/RNA extraction room). The steps in the PCR workflow are always unidirectional, from DNA /RNA extraction to amplification. This principle holds for working procedures as well as for reagents and consumables. Sample DNA/RNA Product

Handling steps Reagents & disposables Storage

Handling steps Reagents & disposables Storage

Handling steps Reagents & disposables Storage

Handling steps Reagents & disposables

Figure1: Critical steps in PCR workflow 2.2 Avoiding one possible source of contamination Screen individual reagents and consumables in PCR before they are used to assay unknowns. This is especially true for oligonucleotides (primers and probes). In addition, you should include a negative control in the PCR run for every step that uses fresh reagents and disposables. It is also helpful to include a reliable positive control to demonstrate true gene expression. General Guidelines for Preventing Cross-contamination The tables below summarize the guidelines for PCR laboratory to prevent PCR contamination Area of Concern All stages Disposables and reagents Use of reagents and disposables is room-specific. Always store a unique set of reagents and disposables in each of the procedure-specific rooms (DNA isolation, PCR, etc.) and use that set only in that room. Use each plastic disposable only once. Divide reagents into aliquots that will be consumed in a single procedure (DNA/RNA isolation, PCR, etc.). make only enough regents for the number of samples you are preparing (e.g., if you usually run 10 samples, make aliquots with enough reagents for 11 reactions). Minimize pipetting steps by using reagents premixes (e.g., PCR master mixes available from Roche Applied Science) whenever possible. Discard any reagents left over from a procedure rather than storing them and STANDARD TECHNICAL MANUAL Steps To Take

3.

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HAEMATOLOGY DEPARTMENT, HOSPITAL UNIVERSITI SAINS MALAYSIA TITLE: PROCEDURE NO. RNA EXTRACTION HUSM/HEMA-UPT/STM-M1
reusing them. Cleaning/ Decontaminating the laboratory environment Regularly wipe pipette tips with ethanol-soaked tissue. Access to the laboratory is controlled and limited. Clean the work place with 70% alcohol before and after used Always use cleaning agents that are suitable for and dedicated to decontaminating PCR lab surfaces and instruments with bleach (make sure this is allowed for your instrument.) or with DNA Remover, DNAZap or DNAExitus Plus. UV irradiation (at 254 nm) can inactivate DNA contamination in disposable, reagents and PCR samples. UV dimerizes that thymidine residues in DNA, thus rendering it incapable of serving as a PCR template. For RNA; all glassware and solutions (where possible) must be prepared with 0.1% DEPC, incubated overnight at room temperature and then autoclaved to inactive the DEPC. Always use ultrapure water for reagent and sample dilution. Do not use autoclaved water because it may be contaminated with DNA if the autoclave is also used for sterilization of cultures. Autoclaving facilities must be completely separate from the PCR labs and steam must not be carried into the PCR labs via the air conditioning system. Always wear gloves and lab coats during the procedures Removed gloves when leaving the lab Always clean lab clothing Never touch any surface of the disposable tubes/ tips that will make contact with the samples Using dedicated pipettes for DNA and RNA process activities Decontaminate the laminar flow cabinet with UV light for 30 minutes before the process Change all waste collectors

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Water

laboratory clothing

PCR sample tube or pipette tips instrument

END OF DOCUMENT
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