For Research Use Only Ver

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1 INSTRUCTION MANUAL Quick-cfRNA Serum & Plasma Kit Catalg N. R1059 Highlights High-quality cell-free RNA is easily and rbustly purified frm up t 3 ml f plasma, serum, r ther bilgical fluids. Purified cell-free RNA is immediately ready fr dwnstream applicatins, including RT-qPCR and Next- Generatin Sequencing. Up t 515x mre micrrna is recvered cmpared t cmparable prducts, enabled by innvative binding system. Cntents Prduct Cntents... 1 Specificatins... 1 Prduct Descriptin & Prcedure Overview... 2 Reagent Preparatin... 3 Prtcl Appendices A. DNase I Treatment... 5 B. Sample Handling and Quantificatin Methds... 5 C. Prcessing High Sample Input Vlume... 6 D. Prcessing Samples in DNA/RNA Shield... 7 E. Cmpatibility with Streck Bld Tubes... 8 Trubleshting... 9 Ordering Infrmatin Fr Research Use Only Ver

2 Page 1 Satisfactin f all Zym Research prducts is guaranteed. If yu are dissatisfied with this prduct, please call Prduct Cntents Quick-cfRNA Serum & Plasma Kit (Kit Size) Prteinase K & Strage Buffer R1059 (50 Preps.) 3 x 20 mg Strage Temperature -20 ºC (after mixing) Quick-cfRNA Digestin Buffer 50 ml Rm Temp. Quick-cfRNA Binding Buffer 100 ml Rm Temp. RNA Recvery Buffer 10 ml Rm Temp. RNA Prep Buffer 50 ml Rm Temp. RNA Wash Buffer (Cncentrate) 12 ml Rm Temp. Spin-Away Filters 50 Rm Temp. 25 ml Reservirs 50 Rm Temp. Zym-Spin IC Clumns 50 Rm Temp. Cllectin Tubes 100 Rm Temp. DNase/RNase-Free Water 4 ml Rm Temp. Instructin Manual 1 - Nte Integrity f kit cmpnents is guaranteed fr up t ne year frm date f purchase. Reagents are rutinely tested n a lt-t-lt basis t ensure they prvide the highest perfrmance and reliability. Specificatins Sample Types 1 ml f plasma, serum, cerebrspinal fluids, amnitic fluids, urine, r ther bilgical fluids. Max vlume f 3 ml input can be prcessed per filter (see Appendix C, Page 6). Ntes: 1 Quantified using flurescence-based technlgy (Qubit 3.0 frm Therm Fisher Scientific) Nte Trademarks f Zym Research Crpratin. This prduct is fr research use nly and shuld be used by trained prfessinals. It is nt intended fr use in diagnstic prcedures. Sme reagents included with this kit are irritants. Wear prtective glves and eye prtectin. Fllw safety guidelines and rules enacted by yur research institutin r facility. Purity High quality cell-free RNA (cfrna) is ready fr all sensitive dwnstream applicatins such as RT-qPCR and Next-Generatin Sequencing. Size RNA 17 nt. Yield Yields are sample dependent and may vary. Typical recvery ranges frm ng/ml f human plasma r serum. 1 Kit is ptimized fr recvery f ttal cfrna, including small RNA and micrrna. Elutin Vlume cfrna can be eluted int 15 µl (t maximize ttal yield) r as little as 6 µl (fr highly cncentrated eluate) f DNase/RNase-Free Water. Prcessing Time Typically requires 30 minutes hands-n time t prcess 10 samples. Sample digestin requires 2 hurs. Required Equipment Micrcentrifuge, vrtex, water bath r heating blck, vacuum, and vacuum manifld. Trademarks f Zym Research Crpratin. This prduct is fr research use nly and shuld be used by trained prfessinals. It is nt intended fr use in diagnstic prcedures. Sme reagents included with this kit are irritants. Wear prtective glves and eye prtectin. Fllw safety guidelines and rules enacted by yur research institutin r facility.

3 Page 2 Prduct Descriptin & Prcedure Overview The Quick-cfRNA Serum & Plasma Kit enables simple and efficient preparatin f ttal cell-free RNA (including prtein-bund, exsmal, micrrna, and ther small RNA) frm serum, plasma, and ther bilgical fluids. The kit des nt use txic phenl-chlrfrm r cumbersme prtein precipitatin and remval steps. Recvered cell-free RNA scales linearly relative t the sample input vlume up t 3 ml. Zym- Spin Technlgy enables islatin f ultra-pure cell-free RNA suitable fr all subsequent analyses, such as RT-qPCR and NGS. Islate up t 515x mre micrrna cmpared t tw separate kits frm supplier Q. Samples frm the same dnr (plasma frm 61y-F) were prcessed using the manufacturers suggested prtcl and eluted in 30 µl. Quantificatin f human mir-16-5p was assayed using the methd described in Busk P. K., BMC Biinfrmatics, The micrrna yield frm the QuickcfRNA Serum & Plasma scales linearly t input vlume. Extractin Kit Zym Supplier Q Reads 1,041, ,153 % reads passing filters 76.6% 58.5% % align t hg % 63.9% % align t mirbase 43.6% 0.4% mirnas detected Obtain rbust cell-free RNA sequencing results. Ttal cell-free RNA was islated frm 200 µl plasma frm fur different dnrs. RealSeq-Bifluids Library Prep Kit (SmaGenics) was used t generate RNA sequencing library and ran n MiniSeq System (Illumina). Averages f reads, read quality, alignments, and micrrna diversity quantificatin are summarized abve. Cellfree RNA islated using Quick-cfRNA Serum & Plasma yielded higher quality reads, better alignment t sequence databases, and achieved recvery f 463 mre micrrna species.

4 Page 3 Ntes: Reagent Preparatin Prir t use, recnstitute lyphilized 20 mg Prteinase K with 1,040 µl Prteinase K Strage Buffer. Vrtex t disslve. Stre at -20 ºC. Add 48 ml 100% ethanl (52 ml 95% ethanl) t the 12 ml RNA Wash Buffer (Cncentrate). Prtcl All centrifugatin steps shuld be perfrmed at 12,000 x g fr 30 secnds in a micrcentrifuge unless specified. All steps shuld be perfrmed at rm temperature (20-30 ºC). If prcessing mre than 1 ml per islatin prcess, please refer t Appendix C fr mre infrmatin. Fr rapid preparatin, see Quick Setup Guide belw. 1 Centrifugatin is necessary fr efficient depletin f residual cells, cell debris, and particulate matter. If cryprecipitates are visible after freeze-thaw cycles, it is recmmended that these are remved by centrifuging again, prir t digestin. Fr recmmended plasma preparatin methds, see Appendix B and references mentined belw: a) Tuck MK, et al. Jurnal f Prteme Research Jan; 8(1): b) Köberle V, et al. PLS One Sep; 8(9): e75184 c) Peter B. Gahan. Circulating Nucleic Acids In Early Diagnsis, Prgnsis and Treatment Mnitring: An Intrductin. pg Centrifuge samples 12,000 x g fr 15 minutes t remve any cell debris and precipitate In a new 15 ml cnical tube, add 200 µl Quick-cfRNA Digestin Buffer per 200 µl f sample (plasma, serum, r ther bilgical fluids) and mix by pipetting. If the input sample vlume is 1.5 ml, use a 50 ml cnical tube. 3. Add 10 µl Prteinase K per 200 µl f sample and mix thrughly by vrtexing fr 10 secnds. Incubate at 37 C fr 2 hurs. 4. Add 1 vlume Quick-cfRNA Binding Buffer t the digested sample and mix thrughly by vrtexing fr 10 secnds. Example: Add 400 µl binding buffer t 410 µl digested mixture. 5. Add 1.5 vlumes 100% isprpanl t the mixture frm Step 4 and mix thrughly by vrtexing fr 10 secnds. Example: Add 1.2 ml f 100% isprpanl t 810 µl sample mixture. Step 1 Quick Setup Guide Sample vlume 200 µl 500 µl 1 ml Quick-cfRNA Digestin Buffer 200 µl 500 µl 1 ml Step 2 Prteinase K 10 µl 25 µl 50 µl Mix thrughly by vrtexing and incubate at 37 C fr 2 hurs Step 3 Quick-cfRNA Binding Buffer 400 µl 1 ml 2 ml Step 4 100% Isprpanl 1.2 ml 3 ml 6 ml 2 The vacuum pump shuld be able t apply at least 400 mmhg pressure. Final mixture vlume 2 ml 5 ml 10 ml 6. Insert 25 ml Reservir int Spin-Away Filter. Place the assembly nt vacuum manifld. 2

5 Page 4 7. Lad the entire mixture int the reservir and turn n the vacuum until the entire mixture has been cmpletely drawn thrugh the Spin-Away Filter. 1 Turn ff the vacuum pump and discard the reservir. 8. Add 600 µl RNA Prep Buffer and turn n the vacuum until all f the liquid cmpletely passes thrugh the Spin-Away Filter. Turn ff the vacuum. Ntes: 1 Fr 3 ml f sample vlume, it may take up t 15 minutes fr the digestin mixture t cmpletely pass thrugh the filter. 9. Transfer the Spin-Away Filter t a Cllectin Tube and centrifuge fr 2 minutes t remve residual buffer. Place the filter int a new micrcentrifuge tube (nt prvided). 10. Add 200 µl RNA Recvery Buffer directly t the Spin-Away Filter. Incubate fr 3 minutes and centrifuge. Save the Flw-Thrugh!!! 11. Add 300 µl ethanl (95-100%) t the flw-thrugh. Mix by pipetting and transfer int a Zym-Spin IC Clumn in a new Cllectin Tube. Centrifuge and discard the flw-thrugh. 12. Add 400 µl RNA Prep Buffer t the clumn. Centrifuge and discard the flwthrugh. 13. Add 700 µl RNA Wash Buffer t the clumn. Centrifuge and discard the flwthrugh. 14. Add 400 µl RNA Wash Buffer t the clumn and centrifuge fr 2 minutes t ensure cmplete remval f the wash buffer. Transfer the clumn int a clean micrcentrifuge tube (nt prvided). 15. Add 15 µl DNase/RNase-Free Water directly t the clumn matrix, incubate fr 2 minutes and centrifuge t elute cfrna. 2 The eluted cfrna can be used immediately r stred at -70 C. 3 2 Alternatively, fr highly cncentrated cfrna, use 6 µl elutin vlume. 3 Quantificatin f cfrna requires highly sensitive assays due t very lw amunts fund in bilgical fluids. Please see Appendix B fr details.

6 Page 5 Ntes: 1 Prir t use, recnstitute the lyphilized DNase I as indicated n the vial. Stre frzen aliquts. Unit definitin ne unit increases the absrbance f a high mlecular weight DNA slutin at a rate f A260 units/min/ml f reactin mixture at 25 C. Appendix A: DNase I Treatment (Optinal) Fr cmplete remval f cfdna frm cfrna eluate, the DNase digestin prcedure can be perfrmed using the DNase I Set (Cat. N. E1010) 1 and the Olig Clean & Cncentratr (Cat. N. D4060, D4061). Fr each sample t be treated, prepare DNase I treatment in RNase-free tube (nt prvided). Mix well by gentle inversin: Eluate vlume adjusted with water r TE buffer 40 µl DNase I 5 µl DNA Digestin Buffer 5 µl Ttal Vlume 50 µl Incubate at rm temperature (20-30 C) fr 15 minutes then start with Step 1 f Olig Clean & Cncentratr prtcl. Appendix B: Sample Handling and Quantificatin Methds Cell-free nucleic acid is prtected frm the denaturing envirnment f bifluids by encapsulatin in extracellular vesicles r binding t prteins. We recmmend minimizing freeze-thaw cycles and t avid vigrus shaking prir t sample digestin t preserve integrity f the prtective vesicles and prteins. Slw thawing f bifluids at 4 C increases the frmatin f precipitate that is hard t digest and may clg clumns. We recmmend thawing plasma samples at rm temperature r 37 C t minimize the frmatin f precipitate. Remve precipitate by centrifuging samples at 12,000 x g fr 15 minutes at rm temperature. Precipitate can cause inefficient digestin, which can lead t filter clgging and lwer yield. Highly sensitive quantificatin methds are needed t accurately measure dilute eluates such as cell-free nucleic acid frm bilgical fluids. We recmmend using flurescence-based detectin methds (e.g. Qubit frm Therm-Fisher) r sensitive electrphresis systems (e.g. TapeStatin r Bianalyzer frm Agilent). If a sample-t-sample variatin cntrl is needed in micrrna RT-qPCR analysis, an exgenus micrrna can be used. We recmmend spiking 1 t 10 pg f an exgenus micrrna (e.g. cel-mir-39-3p micrrna; sequence available at mirbase.rg) t each prep after cmpletin f sample digestin (after Step 2).

7 Page 6 Appendix C: Prcessing High Sample Input Vlume Up t 3 ml The kit is supplied with enugh reagents t prcess up t a max f 1 ml sample input. T prcess all 50 preps with 2 ml sample input, purchase the fllwing additinal buffers: 3 f Prteinase K & Strage Buffer, 20 mg set (Catalg N. D ) 1 f Quick-cfRNA Digestin Buffer, 50 ml (Catalg N. R ) 1 f Quick-cfRNA Binding Buffer, 100 ml (Catalg N. R ) T prcess all 50 preps with 3 ml sample input, purchase the fllwing additinal buffers: 6 f Prteinase K & Strage Buffer, 20 mg set (Catalg N. D ) 2 f Quick-cfRNA Digestin Buffer, 50 ml (Catalg N. R ) 2 f Quick-cfRNA Binding Buffer, 100 ml (Catalg N. R ) Fr rapid preparatin, see Quick Setup Guide belw. Quick Setup Guide Sample vlume 1 ml 2 ml 3 ml Step 1 Quick-cfRNA Digestin Buffer 1 ml 2 ml 3 ml Step 2 Prteinase K 50 µl 100 µl 150 µl Mix thrughly by vrtexing and incubate at 37 C fr 2 hurs Step 3 Quick-cfRNA Binding Buffer 2 ml 4 ml 6 ml Step 4 100% Isprpanl 6 ml 12 ml 18 ml Final mixture vlume 10 ml 20 ml 30 ml Prceed t Step 6 (Page 3) t cntinue islatin prcess. Please refer t Ordering Infrmatin (Page 8) fr rdering details n all kit cmpnents.

8 Page 7 Appendix D: Prcessing Samples in DNA/RNA Shield Fr cell-free bifluid samples stred in DNA/RNA Shield (R1200) at 1:1 rati, perfrm the fllwing mdified Prteinase K digestin. Fr rapid preparatin, please see Quick Setup Guide belw. 1. Add 25 µl Prteinase K per 1 ml samples in DNA/RNA Shield and mix thrughly by vrtexing fr 10 secnds. Incubate at 37 C fr 2 hurs. Example: 500 µl bifluid, 500 µl DNA/RNA Shield (2X cncentrate), 25 µl Prteinase K. 2. Add 1 vlume Quick-cfRNA Digestin Buffer t the digested sample and mix thrughly by vrtexing fr 10 secnds. Example: Add 1 ml Quick-cfRNA Digestin Buffer t 1 ml digested mixture. 3. Add 1 vlume Quick-cfRNA Binding Buffer t the mixture frm Step 2 and mix thrughly by vrtexing fr 10 secnds. Example: Add 2 ml Quick-cfRNA Binding Buffer t 2 ml sample mixture. 4. Add 1.5 vlume 100% isprpanl t the mixture frm Step 3 and mix thrughly by vrtexing fr 10 secnds. Example: Add 6 ml f 100% isprpanl t 4 ml sample mixture. Quick Setup Guide Sample/Shield Mixture Vlume 200 µl 500 µl 1 ml Step 1 Prteinase K 5 µl 12.5 µl 25 µl Mix thrughly by vrtexing and incubate at 37 C fr 2 hurs Step 2 Step 3 Quick-cfRNA Digestin Buffer Quick-cfRNA Binding Buffer 200 µl 500 µl 1 ml 400 µl 1 ml 2 ml Step 4 100% Isprpanl 1.2 ml 3 ml 6 ml Final mixture vlume 2 ml 5 ml 10 ml Prceed t Step 6 (Page 3) t cntinue islatin prcess.

9 Page 8 Appendix E: Cmpatibility with Streck Bld Tubes Plasma/serum that are samples derived frm bld stred in Streck bld tubes cntain chemically fixed prteins. In rder t cmpletely digest samples derived frm Cell-Free RNA BCT frm Streck, plasma/serum samples must be incubated in Quick-cfRNA Digestin Buffer at 55 C prir t prteinase K digestin. Please fllw the prtcl belw fr islatin f cell-free RNA frm plasma/serum samples derived frm Streck Bld Tubes. 1. Centrifuge samples 12,000 x g fr 15 minutes t remve any cell debris and precipitate. 2. In a new 15 ml cnical tube, add 200 µl Quick-cfRNA Digestin Buffer per 200 µl f sample (plasma, serum, r ther bilgical fluids) and mix by pipetting. If the input sample vlume is 1.5 ml, use a 50 ml cnical tube. 3. Incubate the mixture at 55 C fr 30 minutes. Prceed t Step 3 (Page 3) t cntinue t cfrna islatin.

10 Page 9 Trubleshting: Fr Technical Assistance, please cntact r tech@zymresearch.cm Prblem Pssible Causes and Suggested Slutins Sme bilgical fluids, such as plasma, are prtein-rich and require cmplete Prteinase K digestin prir t passing thrugh the Spin- Away Filter. Remving visible precipitate prir t prcessing is recmmended fr ptimal kit perfrmance. Spin-Away Filter Clgging Althugh digesting samples fr 2 hurs at 37 C is sufficient fr mst samples, extending the digestin time t 3-4 hurs may help if samples cntain residual precipitate that is hard t remve. D nt add mre than the indicated amunt f prteinase K D nt increase the digestin temperature abve 37 C. Increased vacuum pressures ( 400 mmhg) can help the lysate flw thrugh the filter faster. Yield can vary significantly frm dnr t dnr. Increased levels f circulating nucleic acids in the bld reflect pathlgical prcesses, including cancers, inflammatry diseases, and trauma (Schwarzenbach H. et al. Nat. Rev. Cancer, 2011). Lw Yield Incmplete digestin can cause clgging f the Spin-Away Filter and can result in inefficient RNA binding. Ensure sample is cmpletely digested befre prceeding t sample binding.

11 Page 10 Ordering Infrmatin Prduct Descriptin Catalg N. Kit size Quick-cfRNA Serum & Plasma Kit R preps Quick-cfDNA/cfDNA Serum & Plasma Kit R preps Quick-cfDNA Serum & Plasma Kit D preps Olig Clean & Cncentratr D4060 D4061 DNase I Set E1010 Set DNA/RNA Shield, 2X cncentrate R R preps 200 preps 25 ml 125 ml Fr Individual Sale Catalg N. Amunt Prteinase K & Strage Buffer D D mg set 20 mg set Quick-cfRNA Digestin Buffer R ml Quick-cfRNA Binding Buffer R ml RNA Recvery Buffer R ml RNA Prep Buffer RNA Wash Buffer (cncentrate) R R R R R R R ml 25 ml 100 ml 6 ml 12 ml 24 ml 48 ml Spin-Away Filters C F ml Reservirs C Zym-Spin IC Clumns Cllectin Tubes DNase/RNase-Free Water C C C C C W W W W W ml 4 ml 6 ml 10 ml 30 ml