Agilent miRNA 1st-Strand cDNA 600036 Synthesis Kit Instruction Manual PDF

miRNA 1st-Strand cDNA Synthesis Kit

Instruction Manual

Catalog #600036 Revision E.0

For Research Use Only. Not for use in diagnostic procedures. 600036-12

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miRNA 1st-Strand cDNA Synthesis Kit

CONTENTS Materials Provided .............................................................................................................................. 1 Storage Conditions .............................................................................................................................. 1 Additional Materials Required .......................................................................................................... 1 Notices to Purchaser ........................................................................................................................... 2 Introduction ......................................................................................................................................... 3

Overview of the miRNA 1st-Strand cDNA Synthesis Protocol ............................................ 3 Preprotocol Considerations ................................................................................................................ 4

Preparation of Total RNA ..................................................................................................... 4 Quantity of RNA to Use as Template .................................................................................... 4 Use of Manganese Chloride .................................................................................................. 4 No-PAP Control .................................................................................................................... 4 QPCR Forward Primer Selection .......................................................................................... 5 General Notes ........................................................................................................................ 5

Protocols ............................................................................................................................................... 6 Polyadenylation Reaction ...................................................................................................... 6 RNA Purification ................................................................................................................... 7 1st-Strand cDNA Synthesis ................................................................................................... 9

Troubleshooting ................................................................................................................................ 10 References .......................................................................................................................................... 10 Endnotes ............................................................................................................................................. 10 MSDS Information ............................................................................................................................ 10

miRNA 1st-Strand cDNA Synthesis Kit 1

miRNA 1st-Strand cDNA Synthesis Kit

MATERIALS PROVIDED

Catalog #600036 Materials Provided Concentration Quantity

E. coli Poly A Polymerase (PAP) 2 U/l 100 U

E. coli Poly A Polymerase Buffer 5 200 l

Manganese chloride 25 mM 50 l

rATP 10 mM 250 l

Glycogen 20 mg/ml 25 l

AffinityScript RT Buffer 10 100 l

RT Adaptor Primer 10 M 50 l

100 mM dNTP mix 25 mM each 40 l

AffinityScript RT/RNase Block Enzyme Mixture 50 l

Universal Reverse Primer 3.125 M 200 l a The kit provides sufficient reagents for fifty 20-l polyadenylation reactions and fifty 20-l cDNA synthesis

reactions.

STORAGE CONDITIONS All materials: Store at 20C upon receipt.

ADDITIONAL MATERIALS REQUIRED Nuclease-free water

Revision E.0 Agilent Technologies, Inc. 2015.

2 miRNA 1st-Strand cDNA Synthesis Kit

NOTICES TO PURCHASER This product is licensed for research and development only under patents and patent applications jointly owned by BIOTIUM and ALLELOGIC.

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155, 5,677,152 (claims 1 to 23 only), 5,773,258 (claims 1 and 6 only), 5,407,800, 5,322,770, 5,310,652, 5,994,056, 6,171,785, and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchasers own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Notice to Purchaser: Limited License Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchasers own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

miRNA 1st-Strand cDNA Synthesis Kit 3

INTRODUCTION The miRNA 1st-Strand cDNA Synthesis Kit provides the reagents to elongate miRNAs in a polyadenylation reaction and then reverse transcribe the polyadenylated RNA into QPCR-ready cDNA. The cDNA may then be amplified using the provided universal reverse primer and a unique forward primer that is specific to the miRNA target of interst (not provided).

Overview of the miRNA 1st-Strand cDNA Synthesis Protocol

Elongation of miRNA with Poly A Polymerase Because of their short length, miRNAs are difficult to detect with standard QRT-PCR protocols. To elongate the miRNAs, total RNA is first treated with E. coli poly A polymerase (PAP) to generate a poly-A tail at the 3 end of each RNA molecule. The miRNA 1st-strand cDNA synthesis kit provides all the reagents needed for the polyadenylation reaction. Manganese chloride is also included in the cDNA synthesis kit as an optional reagent in the polyadenylation reaction. Adding MnCl2 to the reaction may improve the efficiency of poly A polymerase (see Use of Manganese Chloride in Preprotocol Considerations for further information). If MnCl2 is included, the polyadenylated RNA will need to be purified with a phenol-chloroform extraction and ethanol precipitation before the cDNA synthesis step. The 20 mg/ml stock of glycogen is provided in the kit for this purpose.

Synthesis of 1st-Strand cDNA Following polyadenylation, the RNA is used as template to synthesize 1st-strand cDNA. The cDNA synthesis protocol is optimized for reverse transcription of mRNA and miRNA templates. The reverse transcriptase (RT) provided with the 1st-strand cDNA synthesis kit is Agilent's AffinityScript RT, a genetically engineered version of Moloney murine leukemia virus RT. AffinityScript RT is provided in combination with RNase block as a safeguard against contaminating RNases, and is stringently quality-controlled to verify the absence of nuclease contaminants that adversely affect cDNA synthesis, particularly from small input RNAs. The cDNA synthesis reaction is primed using the RT adaptor primer. This carefully designed primer anneals to the 3 poly-A tail that was added during the polyadenylation reaction. In addition to this poly-A binding sequence, the RT adaptor primer also contains additional bases that create a universal sequence tag on each cDNA strand that is synthesized. This universal tag is incorporated at the 5 end of the cDNA.

4 miRNA 1st-Strand cDNA Synthesis Kit

PREPROTOCOL CONSIDERATIONS

Preparation of Total RNA The miRNA 1st-strand cDNA synthesis kit uses total RNA as the starting material in the protocol. This RNA sample may be prepared from any source or cell type using most standard RNA purification procedures. In some cases, the RNA isolation protocol may need to be modified to ensure recovery of small RNAs. The quality of the RNA preparation may impact the sensitivity of miRNA detection. RNA samples with OD260/280 ratios of 1.82.0 are optimally pure. Enriching the RNA sample for miRNA is not necessary, but may improve detection of some difficult targets.

Quantity of RNA to Use as Template The miRNA 1st-strand cDNA synthesis kit can accommodate a range of total RNA input amounts from 30 ng to 1 g. The optimal quantity of RNA template depends on the RNA purity and the expression level of the particular miRNA of interest.

Use of Manganese Chloride Manganese chloride is provided in the miRNA 1st-strand cDNA synthesis kit as an optional reagent in the polyadenylation reaction. Adding MnCl2 to the reaction may improve the efficiency of the poly A polymerase enzyme. However, the presence of MnCl2 during the subsequent cDNA synthesis and PCR reactions could lead to errors in nucleotide incorporation, creating mutations in the DNA sequence.1, 2 In order to prevent MnCl2 from interfering with these downstream steps, if MnCl2 is included in the polyadenylation reaction, purify the polyadenylated RNA with a phenol- chloroform extraction and ethanol precipitation to remove the MnCl2 before cDNA synthesis. A protocol is provided under RNA Purification in the Protocols section of the manual. If MnCl2 is omitted, skip the purification protocol and proceed directly to the 1st-Strand cDNA Synthesis section following polyadenylation.

No-PAP Control To screen for contamination, consider including with the QPCR reactions a no-PAP control cDNA template. The no-PAP control cDNA is prepared from a polyadenylation reaction in which the poly A polymerase is omitted.

miRNA 1st-Strand cDNA Synthesis Kit 5

QPCR Forward Primer Selection The primers used to perform QPCR with the miRNA-derived cDNA are the universal reverse primer provided in the miRNA 1st-strand cDNA synthesis kit and a unique forward primer that allows specific amplification of the target of interest. The universal reverse primer anneals to the cDNA sequence tag that was added to the 5 end of all cDNA species by the RT adaptor primer during 1st-strand cDNA synthesis. For the unique forward primer, use a custom primer specific to the target of interest. Ensure the custom primer is designed to be complementary to the 3 end of the cDNA strand. Generally, the forward primer should be identical in sequence and length to the miRNA itself. Further guidelines on designing a custom forward primer for miRNA detection are available online at www.genomics.agilent.com/files/LitItems/miRNA_primer_design_guidelines .pdf. Once the sequence of the forward primer has been determined, the primer should be ordered through a custom oligo supplier. Dilute the primer stock to a concentration of 3.125 M in TE (5 mM Tris-HCl, pH 8.0 and 0.1 mM EDTA) and store at 20C.

General Notes

Preventing Cross-Contamination Take precautions to minimize the potential for carryover of nucleic acids from one experiment to the next. Use separate work areas and pipettors for pre- and post-amplification steps. Use positive displacement pipets or aerosol-resistant pipet tips.

Preparing a Master Mix for Multiple Samples If analyzing multiple samples, a master mix of reaction components may be prepared by combining the desired multiple of each component. Using a master mix facilitates accurate dispensing of reagents, minimizes loss of reagents during pipetting, and makes repeated dispensing of each reagent unnecessary, all of which help minimize sample-to-sample variation.

Mixing and Pipetting Enzymes Enzymes (e.g. poly A polymerase) should be mixed gently without generating bubbles. Pipet the enzymes carefully and slowly; otherwise, the viscosity of the 50% glycerol in the buffer can lead to pipetting errors.

6 miRNA 1st-Strand cDNA Synthesis Kit

PROTOCOLS

Polyadenylation Reaction

1. Prepare the polyadenylation reactions by adding the following components in order to separate RNase-free 0.5-ml microcentrifuge tubes:

RNase-free water to bring final volume to 20 l (including polymerase added in step 2)

4.0 l of 5 poly A polymerase buffer 1.0 l of rATP (10 mM) 1.0 l of 25 mM MnCl2 (optional) x l of total RNA (30 ng 1 g)

Note Including MnCl2 in the reaction may improve the efficiency of the PAP enzyme. If MnCl2 is included, the polyadenylated RNA will need to be purified with a phenol-chloroform extraction and ethanol precipitation before cDNA synthesis.

2. Add 1 l of E. coli poly A polymerase to each reaction and mix gently (do not vortex). Briefly centrifuge the reactions to collect the contents at the bottoms of the tubes.

3. Incubate the reactions at 37C for 30 minutes.

4. Incubate the reactions at 95C for 5 minutes to terminate adenylation, then immediately transfer the tubes to ice.

5. Proceed to either RNA Purification (if MnCl2 was included) or 1st-Strand cDNA Synthesis (if MnCl2 was omitted), or store the reactions at 20C. For long term storage, store the reactions at 80C.

miRNA 1st-Strand cDNA Synthesis Kit 7

RNA Purification

Note If MnCl2 was included in the polyadenylation reaction, the RNA needs to be purified to remove the MnCl2 before proceeding to cDNA synthesis. Follow the protocol below to purify the RNA with a phenol-chloroform extraction and ethanol precipitation. Alternatively, RNA may be purified by gel filtration using a ready-to-use chromatography column packed with a polyacrylamide gel matrix. When using a chromatography column, the volume of the column filtrate may need to be reduced down to 10 l by drying the filtrate in a vacuum centrifuge.

If MnCl2 was omitted, skip the RNA purification step and proceed directly to the 1st-Strand cDNA Synthesis section.

Additional Materials Required

The RNA purification protocol requires the following materials not included with the miRNA 1st-strand cDNA synthesis kit:

Siliconized 1.5-ml microcentrifuge tubes, RNase-free Phenol:chloroform:isoamyl alcohol [25:24:1 (v/v/v)] Chloroform 3.0 M Sodium acetate, pH 5.2 95% Ethanol prepared with RNase-free water (bring to 4C before use) 80% Ethanol prepared with RNase-free water (bring to 4C before use) Vacuum centrifuge (e.g. SpeedVac concentrator) RNase-free water

Phenol-Chloroform Extraction and Ethanol Precipitation

1. To each polyadenylation reaction sample, add 80 l of RNase-free water. Mix well.

2. Transfer each sample into a siliconized 1.5-ml microcentrifuge tube.

Note The use of low-retention, siliconized tubes helps prevent the loss of small RNAs during the purification procedure.

3. Add 100 l of phenol:chloroform:isoamyl alcohol to each sample and vortex.

4. Spin the samples in a microcentrifuge at 14,000 g for 10 minutes at 4C.

5. For each sample, transfer the aqueous upper-phase (containing the RNA) into a fresh 1.5-ml siliconized tube. Be careful to avoid disturbing the interface between the two layers.

6. Add an equal volume of chloroform to each sample and vortex the mixture.

8 miRNA 1st-Strand cDNA Synthesis Kit

7. Spin the samples in a microcentrifuge at 14,000 g for 10 minutes at 4C.

8. Again, transfer the aqueous upper-phase (containing the RNA) of each sample into a fresh 1.5-ml siliconized tube. Be careful to avoid disturbing the interface between the two layers.

9. Add the following to each sample:

0.5 l of 20 mg/ml glycogen 10 l of 3.0 M NaOAc, pH 5.2

10. Invert the tubes to mix contents then add 300 l of cold 95% ethanol to each sample to precipitate the RNA.

Note Allowing the RNA to precipitate overnight at 20C may improve yield.

11. Spin the samples in a microcentrifuge at 14,000 g for 10 minutes at 4C to pellet the RNA precipitate. A white pellet should be visible near the bottom of the tube following centrifugation.

12. Carefully decant the supernatant without disturbing the pellet.

13. Wash the RNA pellet by adding 300 l of cold 80% ethanol to each tube. Invert the tubes to mix contents.

14. Spin the samples in a microcentrifuge at 14,000 g for 10 minutes at 4C to pellet the washed RNA precipitate. A white pellet should be visible near the bottom of the tube.

15. Carefully decant the supernatant without disturbing the pellet

16. Dry the RNA pellets under vacuum centrifugation for 25 minutes. Do not overdry the sample.

17. Resuspend each RNA pellet in 10 l of RNase-free water.

18. If proceeding directly to 1st-Strand cDNA Synthesis, keep the tubes on ice. For long term storage, store the RNA at 80C.

miRNA 1st-Strand cDNA Synthesis Kit 9

1st-Strand cDNA Synthesis

1. For each RNA sample, prepare a cDNA synthesis reaction by adding the following components in order to a RNase-free microcentrifuge tube:

RNase-free water to bring final volume to 20 l 2.0 l of 10 AffinityScript RT buffer 10 l of polyadenylated and purified RNA

or 4 l of the polyadenylation reaction 0.8 l of dNTP mix (100 mM) 1.0 l of RT adaptor primer (10 M) 1.0 l of AffinityScript RT/RNase Block enzyme mixture

2. Gently mix the reactions (do not vortex) and briefly centrifuge the tubes.

3. Incubate the reactions at 55C for 5 minutes.

4. Transfer the reactions to 25C and incubate for 15 minutes.

5. Transfer the reactions to 42C and incubate for 30 minutes to allow reverse transcription of 1st-strand cDNA.

6. Incubate the reactions at 95C for 5 minutes to terminate reverse transcription.

7. Add up to 280 l of RNase-free water to each reaction.

8. Place the completed 1st-strand cDNA synthesis reactions on ice for immediate use in QPCR. For long-term storage, keep the reactions at 20C.

10 miRNA 1st-Strand cDNA Synthesis Kit

TROUBLESHOOTING Observation Suggestion

No or low yield of 1st-strand cDNA

Ensure the correct stock of dNTP mix (100 mM) was added to the cDNA synthesis reaction.

Increase the concentration of polyadenylated template RNA added to the cDNA synthesis reaction.

If MnCl2 was not included in the polyadenylation reaction, try adding it to improve the efficiency of the reaction.

As a positive control, carry out the entire protocol using the Agilent HeLa-S3 Cell Line Total RNA as the input RNA sample.

REFERENCES 1. Beckman, R. A., Mildvan, A. S. and Loeb, L. A. (1985) Biochemistry 24(21):5810-7. 2. Leung, D. W. (1989) Journal of Methods in Cell and Molecular Biology 1(1):11-15. 3. Kwok, S. and Higuchi, R. (1989) Nature 339(6221):237-8.

ENDNOTES SpeedVac is a registered trademark of Savant Instruments, Inc.

MSDS INFORMATION Material Safety Data Sheets (MSDSs) are provided online at http://www.genomics.agilent.com. MSDS documents are not included with product shipments.

miRNA 1st-Strand cDNA Synthesis Kit Catalog #600036

QUICK-REFERENCE PROTOCOL

Polyadenylation Reaction

1. Prepare the polyadenylation reactions by adding the following components in order to separate RNase-free 0.5-ml microcentrifuge tubes:

RNase-free water to bring final volume to 20 l (including polymerase added in step 2) 4.0 l of 5 poly A polymerase buffer 1.0 l of rATP (10 mM) 1.0 l of 25 mM MnCl2 (optional) x l of total RNA (30 ng1 g)

2. Add 1 l of E. coli poly A polymerase to each reaction and mix gently (do not vortex).

3. Incubate the reactions at 37C for 30 minutes.

4. Incubate the reactions at 95C for 5 minutes to terminate adenylation, then immediately transfer the tubes to ice. To store the reactions, keep the tubes at 20C or 80C.

5. If MnCl2 was included in the reaction, purify the RNA with a phenol-chloroform extraction and ethanol precipitation before proceeding to cDNA synthesis. A protocol for RNA purification is provided in the Protocols section of the manual.

cDNA Synthesis

1. For each RNA sample, prepare a cDNA synthesis reaction by adding the following components in order to a RNase-free microcentrifuge tube:

RNase-free water to bring final volume to 20 l 2.0 l of 10 RT buffer 10 l of polyadenylated and purified RNA or 4 l of polyadenylation reaction 0.8 l of 100 mM dNTP mix (from the 1st-Strand cDNA Synthesis Kit) 1.0 l of RT adaptor primer (10 M) 1.0 l of AffinityScript RT/RNase Block

2. Gently mix the reactions (do not vortex) and incubate at 55C for 5 minutes.

3. Transfer the reactions to 25C and incubate for 15 minutes.

4. Transfer the reactions to 42C and incubate for 30 minutes to allow reverse transcription.

5. Incubate the reactions at 95C for 5 minutes to terminate reverse transcription.

6. Add 280 l of RNase-free water to each reaction. Proceed to QPCR or store at 20C.