Name: retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens
Uploaded: 2018-05-05T13:00:00
Description: Watch this Scientific Journal Video about Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens at JoVE.com

Vi tackar Dr. Thomas Tuschl, chef för laboratoriet för RNA molekylärbiologi, samt medlemmar i hans laboratorium för deras stöd och för att dela den teknik som utvecklats i hans laboratorium och ger tillgång till rörledningen RNAworld. Vi tackar också Dr. Markus Hafner för att dela hans protokoll och ge detaljerade beskrivningar på alla biokemiska och enzymatiska steg som används i hans ursprungliga förfarandet.

1. beredning av alla reagenser och grundfärger
<ol><li>Beställ alla primers och adaptrar som beskrivs i figur 1.</li>
	<li>
Förbereda kalibratorn cocktail materiel, som kommer att bli spetsade i varje enskild ligering.
	<ol>
<li>Återsuspendera den bärare oligonukleotiden (figur 1) till 0,5 µM med RNase-gratis vatten. Återsuspendera de 10 kalibrator oligonukleotider (figur 1) till 100 µM med 0,5 µM carrier...

<ol>
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Histopathol. 26, 797-810 (2011).</li><li>Kakimoto, Y., Kamiguchi, H., Ochiai, E., Satoh, F., Osawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Stability+in+Postmortem+FFPE+Tissues:+Quantitative+Analysis+Using+Autoptic+Samples+from+Acute+Myocardial+Infarction+Patients.">MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients.</a> PLoS One. 10 (6), e0129338 (2015).</li><li>Giricz, O., Reynolds, P. A., Ramnauth, A., Liu, C., Wang, T., Stead, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsa-miR-375+is+differentially+expressed+during+breast+lobular+neoplasia+and+promotes+loss+of+mammary+acinar+polarity.">Hsa-miR-375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity.</a> J. Pathol. 226, 108-119 (2012).</li><li>Hui, A. B., Shi, W., Boutros, P. C., Miller, N., Pintilie, M., Fyles, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+global+micro-RNA+profiling+with+formalin-fixed+paraffin-embedded+breast+cancer+tissues.">Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues.</a> Lab. Invest. 89, 597-606 (2009).</li><li>Zhang, X., Chen, J., Radcliffe, T., Lebrun, D. P., Tron, V. A., Feilotter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+array-based+analysis+of+microRNA+expression+comparing+matched+frozen+and+formalin-fixed+paraffin-embedded+human+tissue+samples.">An array-based analysis of microRNA expression comparing matched frozen and formalin-fixed paraffin-embedded human tissue samples.</a> J. Mol. Diagn. 10, 513-519 (2008).</li><li>Kolbert, C. P., Feddersen, R. M., Rakhshan, F., Grill, D. E., Simon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-platform+analysis+of+microRNA+expression+measurements+in+RNA+from+fresh+frozen+and+FFPE+tissues.">Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues.</a> PLoS One. 8, e52517 (2013).</li><li>Meng, W., McElroy, J. P., Volinia, S., Palatini, J., Warner, S., Ayers, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+microRNA+deep+sequencing+of+matched+formalin-fixed+paraffin-embedded+and+fresh+frozen+cancer+tissues.">Comparison of microRNA deep sequencing of matched formalin-fixed paraffin-embedded and fresh frozen cancer tissues.</a> PLoS One. 8 (5), e64393 (2013).</li><li>Liu, A., Tetzlaff, M. T., Vanbelle, P., Elder, D., Feldman, M., Tobias, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiling+outperforms+mRNA+expression+profiling+in+formalin-fixed+paraffin-embedded+tissues.">MicroRNA expression profiling outperforms mRNA expression profiling in formalin-fixed paraffin-embedded tissues.</a> Int. J. Clin. Exp. Pathol. 2, 519-527 (2009).</li><li>Dijkstra, J. R., Mekenkamp, L. J., Teerenstra, S., De Krijger, I., Nagtegaal, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+in+formalin-fixed+paraffin+embedded+tissue+using+real+time+quantitative+PCR:+the+strengths+and+pitfalls.">MicroRNA expression in formalin-fixed paraffin embedded tissue using real time quantitative PCR: the strengths and pitfalls.</a> J Cell Mol Med. 16, 683-690 (2012).</li><li>Siebolts, U., Varnholt, H., Drebber, U., Dienes, H. P., Wickenhauser, C., Odenthal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissues+from+routine+pathology+archives+are+suitable+for+microRNA+analyses+by+quantitative+PCR.">Tissues from routine pathology archives are suitable for microRNA analyses by quantitative PCR.</a> J. Clin. Pathol. 62, 84-88 (2009).</li><li>Jang, J. S., Simon, V. A., Feddersen, R. M., Rakhshan, F., Schultz, D. A., Zschunke, M. A., Lingle, W. L., Kolbert, C. P., Jen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+miRNA+expression+analysis+using+fluidigm+microfluidics+dynamic+arrays.">Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.</a> BMC Genomics. 12, 144 (2011).</li><li>Andreasen, D., Fog, J. U., Biggs, W., Salomon, J., Dahslveen, I. K., Baker, A., Mouritzen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+microRNA+quantification+in+total+RNA+from+clinical+samples.">Improved microRNA quantification in total RNA from clinical samples.</a> Methods. 50 (4), S6-S9 (2010).</li><li>Kelly, A. D., Hill, K. E., Correll, M., Wang, Y. E., Rubio, R., Duan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+and+microarray-based+interrogation+of+microRNAs+from+formalin-fixed,+paraffin-embedded+tissue:+preliminary+assessment+of+cross-platform+concordance.">Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: preliminary assessment of cross-platform concordance.</a> Genomics. 102, 8-14 (2013).</li><li>Visone, R., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNAs+and+cancer.">MiRNAs and cancer.</a> Am. J. Pathol. 174, 1131-1138 (2009).</li><li>Lu, J., Getz, G., Miska, E. A., Alvarez-Saavedra, E., Lamb, J., Peck, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiles+classify+human+cancers.">MicroRNA expression profiles classify human cancers.</a> Nature. 435, 834-838 (2005).</li><li>Cho, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+potential+biomarkers+for+cancer+diagnosis,+prognosis+and+targets+for+therapy.">MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy.</a> Int. J. Biochem. Cell. Biol. 42, 1273-1281 (2010).</li><li>Hui, A., How, C., Ito, E., Liu, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-RNAs+as+diagnostic+or+prognostic+markers+in+human+epithelial+malignancies.">Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies.</a> BMC Cancer. 11, 500 (2011).</li><li>Tam, S., de Borja, R., Tsao, M. S., McPherson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+global+microRNA+expression+profiling+using+next-generation+sequencing+technologies.">Robust global microRNA expression profiling using next-generation sequencing technologies.</a> Lab. Invest. 94, 350-358 (2014).</li><li>Loudig, O., Wan, T., Ye, K., Lin, J., Wang, Y., Ramnauth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+Adaptation+of+a+Laboratory+Based+cDNA+Library+Preparation+Protocol+for+Retrospective+Sequencing+of+Archived+MicroRNAs+from+up+to+35-Year-Old+Clinical+FFPE+Specimens.">Evaluation and Adaptation of a Laboratory Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.</a> Int J Mol Sci. 18, e627 (2017).</li><li>Kotorashvili, A., Ramnauth, A., Liu, C., Lin, J., Ye, K., Kim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+DNA/RNA+co-extraction+for+analysis+of+microRNAs,+mRNAs,+and+genomic+DNA+from+formalin-fixed+paraffin-embedded+specimens.">Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.</a> PLoS One. 7 (4), e34683 (2012).</li><li>Hafner, M., Renwick, N., Farazi, T. A., Mihailovi&#263;, A., Pena, J. T., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoded+cDNA+library+preparation+for+small+RNA+profiling+by+next-generation+sequencing.">Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing.</a> Methods. 58 (2), 164-170 (2012).</li><li>Loudig, O., Brandwein-Gensler, M., Kim, R. 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Ett mycket reproducerbara och robust cDNA bibliotek förberedelse protokoll för NGS av små RNAs arkiveras i FFPE RNA exemplar presenteras i detta protokoll, som är en modifierad och optimerad version av det förfarande som beskrivs av Hafner o.a. 22
Alla steg i detta protokoll har optimerats för användning med äldre arkiverade och komprometterad total-RNA återhämtat sig från FFPE exemplar. Det viktigaste steget i detta protokoll, för bearbetning av sm...

MicroRNA är anmärkningsvärt väl bevarad i formalin-fast paraffin-inbäddat (FFPE) exemplar1,2,3. Tidigare arbete har visat att uttrycket av dessa kort reglerande icke-kodande enda stranded RNA-molekyler kan utvärderas framgångsrikt använder total-RNA från FFPE prover och tillhandahålla relevanta gen uttryck data jämfört med det ursprungliga färskt vävnaderna4,5,6,7,8. Jämfört med stora messenger RNAs, som har visat sig påverkas kritiskt av FFPE vävnad behandling (formaldehyd, värme, uttorkning, etc.), endogena RNaser och ålder av exemplar, den lilla storleken på MicroRNA (~ 18 – 24 nukleotider) visas att göra dem resistenta mot nedbrytning och motståndskraftig till långsiktig lagring, också visat genom miRNA uttryck studier som överträffar högt dataflöde mRNA studier i arkiverade exemplar9. miRNA uttryck studier med arkiverade kliniska prover, som har mestadels utförts i småskaliga analyser, har visat att enstaka eller multiplexed kvantitativa PCR-analyser, olika typer av microarray technologies, och mest nyligen NGS kan användas för att utvärdera uttrycket av bevarade MicroRNA efter optimering av dessa analyser10,11,12,13,14.
Med tanke på att dysreglering av miRNA uttryck har associerats med utvecklingen av en mängd mänskliga maligniteter och att det finns potentiellt ett enormt utbud av kliniskt kommenterad arkiverade exemplar, det har blivit uppenbart att dessa små RNA molekyler utgör en lovande potentiell cancer biomarkörer15,16,17,18. Användning av en hög genomströmning gen uttryck teknik såsom NGS har fördelen av att ge en global utvärdering av alla miRNA avskrifter jämfört med riktade teknik såsom PCR eller microarrays19. Av denna anledning, var en optimerad, prisvärt och enkelt tillämpliga protokoll för cDNA bibliotek beredning av små RNAs från äldre arkiverade prover för NGS optimerad för att möjliggöra storskalig retrospektiva studier20.
Tidigare etablerade vi ett samtidiga RNA/DNA extraktion protokoll för separat återvinning av RNA och DNA från äldre arkiverade exemplar, som vi hittade för att överträffa modern kommersiella kit21. Använder detta extraktion protokoll, för att få total-RNA från FFPE-vävnad arkiveras under längre tider, optimerat vi utarbetandet av cDNA bibliotek för NGS av MicroRNA bevaras i kliniska prover för upp till 35 år. Dessutom i en nyligen publicerad studie var vi beredda cDNA bibliotek från kliniskt sekretessbelagda ductal carcinoma i situ (DCIS) exemplar, identifierade vi Differentiellt uttryckta MicroRNA som validerades av kvantitativa PCR, som anges att specifika miRNA uttryck förändringar kan upptäckas i DCIS lesioner från patienter som utvecklar bröstcancer jämfört med DCIS lesioner från patienter som inte utvecklar bröstcancer.
Med tanke på kostnaden för kommersiella kit för beredning av små RNA cDNA bibliotek, potentialen för deras utsättning, samt användningen av upphovsrätt/patentskyddade reagenser som inte kan optimeras, beslutade vi att anpassa en tidigare publicerade laboratoriebaserade och kit-gratis 3' barcoded cDNA bibliotek förberedelse protocol for NGS av små RNAs arkiveras i FFPE exemplar som möjliggör samtidig analys av 18 prover22. Detta protokoll ger en idealisk och robust stegvisa förfarande med visuella och tekniska utvärderingen kontrollpunkter, som var kritiska för anpassning till FFPE RNA exemplar, och har en stor tillväxtpotential för att andra källor till komprometterad eller svårt att använda RNA material. Ursprungliga protokollets tillämpning förbättrades genom att ersätta radioaktivt märkta storlek markörer med lysrör (t.ex., SYBR guld) detekterbara RNA storlek-markörer som används vid urval av sammanskrivna bibliotek på stora polyakrylamidgeler. Optimerad protokollet bygger på ligering av 3' barcoded adaptrar för 18 enskilda FFPE RNA exemplar, som utgör då poolade tillsammans för att genomgå 5' adapter ligatur, omvänd-Transkription, och en pilot PCR analys för skräddarsydda förstärkning av den slutliga cDNA bibliotek före storskaliga PCR-amplifiering, rening och NGS på en hög genomströmning sequencer.

<table>
	<tbody>
		<tr>
			<td>1% Triton x-100</td>
			<td>Invitrogen</td>
			<td>HFH10</td>
			<td></td>
		</tr>
		<tr>
			<td>10mM ATP</td>
			<td>Ambion</td>
			<td>AM8110G</td>
			<td></td>
		</tr>
		<tr>
			<td>10X dNTPs</td>
			<td>Ambion</td>
			<td>AM8110G</td>
			<td></td>
		</tr>
		<tr>
			<td>10x TBE</td>
			<td>Thermofisher Scientific</td>
			<td>15581044</td>
			<td></td>
		</tr>
		<tr>
			<td>14M Mercaptoethanol</td>
			<td>Sigma</td>
			<td>O3445I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>20 nt ladder</td>
			<td>Jena Bioscience</td>
			<td>M-232S</td>
			<td></td>
		</tr>
		<tr>
			<td>20mg/ml Bovine Serum Albumine</td>
			<td>Sigma</td>
			<td>B8894-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>50X Titanium Taq</td>
			<td>Clontech Laboratories</td>
			<td>639208</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Fisher Scientific</td>
			<td>7727-54-0</td>
			<td></td>
		</tr>
		<tr>
			<td>BRL Vertical Gel Electrophoresis System with glass plates and combs</td>
			<td>GIBCO</td>
			<td>V16</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D9170-5VL</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf microcentrifuge 5424R</td>
			<td>USA scientific</td>
			<td>4054-4537Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppndorf Thermomixer</td>
			<td>USA scientific</td>
			<td>4053-8223Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Siliconized Low-Retention Microcentrifuge Tubes 1.5ml</td>
			<td>Fisher Scientific</td>
			<td>02-681-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Breaker Tube 0.5 ml</td>
			<td>IST Engineering Inc,</td>
			<td>3388-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus 7cm x10cm- Mini-sub Cell GT with gel trays and combs</td>
			<td>Biorad</td>
			<td>1704446</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycoblue</td>
			<td>Ambion</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Jersey-Cote</td>
			<td>LabScientific, Inc</td>
			<td>1188</td>
			<td></td>
		</tr>
		<tr>
			<td>KcL 2M</td>
			<td>Ambion</td>
			<td>AM9640G</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 1M</td>
			<td>Ambion</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Minifuge dual rotor personal centrifuge</td>
			<td>USA scientific</td>
			<td>2641-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Model V16 polyacrylamide gel electrophoresis apparatus, glasses, combs, and spacers</td>
			<td>Ciore Life Science</td>
			<td>21070010</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td>IDT</td>
			<td>Defined during order</td>
			<td></td>
		</tr>
		<tr>
			<td>Owl EasyCast B2 mini electrophoresis system- with gel trays and combs</td>
			<td>Thermofisher Scientific</td>
			<td>B2</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiaquick Gel Extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzyme PmeI</td>
			<td>NEB</td>
			<td>R0560S</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe Imager 2.0</td>
			<td>Life Technologies</td>
			<td>G6600</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe Imager 2.0 blue light transilluminator</td>
			<td>Thermofisher</td>
			<td>G6600</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE agarose</td>
			<td>Lonza</td>
			<td>50002</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III reverse transcription kit</td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td></td>
		</tr>
		<tr>
			<td>SybrGold</td>
			<td>Life Technologies</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 1</td>
			<td>NEB</td>
			<td>M0204S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 2 Truncated K227Q</td>
			<td>NEB</td>
			<td>0351L</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Fisher Scientific</td>
			<td>O3446I-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Themocycler with heated lid</td>
			<td>Applied Biosystem</td>
			<td>4359659</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris 1M pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567027</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris 1M pH8.0</td>
			<td>Ambion</td>
			<td>AM9855G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Sequagel system concentrate, diluent, and buffer</td>
			<td>National Diagnostics</td>
			<td>EC-833</td>
			<td></td>
		</tr>
	</tbody>
</table>

retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens

– Arkiveras, kliniskt klassificeras formalin-fast kan paraffin-inbäddat (FFPE) vävnader ge nukleinsyror för retrospektiv molekylära studier av cancerutveckling. Med hjälp av icke-invasiva eller pre-maligna lesioner från patienter som senare utvecklar invasiv sjukdom, kan gen uttryck analyser identifiera tidiga molekylära förändringar som predisponerar för cancerrisk. Det har beskrivits väl att nukleinsyror återhämtat sig från FFPE vävnader har genomgått allvarliga fysiska skador och kemiska modifieringar, vilket försvårar deras analys och allmänt kräver anpassade analyser. MikroRNA (Mirna), dock som föreställer en liten klass av RNA-molekyler som spänner endast upp till ~ 18 – 24 nukleotider, har visat sig tåla långvarig lagring och framgångsrikt har analyserats i FFPE prover. Här presenterar vi en 3' barcoded kompletterande DNA (cDNA) bibliotek förberedelse protokoll specifikt optimerad för analys av små RNAs utvinns ur arkiverade vävnader, som visades nyligen för att vara robust och mycket reproducerbara när använda arkiveras kliniska prover lagras i upp till 35 år. Detta bibliotek preparat är väl anpassade till multiplex analysen av nedsatt/försämrad material där RNA prover (upp till 18) är sammanskrivna med enskilda 3' barcoded adaptrar och sedan poolade tillsammans för efterföljande enzymatisk och biokemiska preparat före analys. Alla reningar utförs av polyakrylamid gelelektrofores (sidan), som tillåter storlek-specifika val och rikedomar av barcoded små RNA arter. Detta cDNA bibliotek preparat är väl anpassade till minuten RNA ingångar, som en pilot polymeras-kedjereaktion (PCR) tillåter bestämning av en specifik amplifiering cykel att producera optimala mängder material för nästa generations sekvensering (NGS). Detta tillvägagångssätt var optimerad för användning av försämrade FFPE RNA från prover som sparas i upp till 35 år och ger mycket reproducerbara NGS data.

Som beskrivs i metoden här, sammanlagt 18 enskilda FFPE RNA-prover (100 ng varje) ställs in i separat rör genomgå 3' adenylated barcoded oligonukleotiden T4 ligering över natten. Nästa dag, är de enzymatiska reaktionerna värme-avaktiveras, kombineras och fälls ut i ett enda rör. RNA pelleten är resuspended och de ligated RNA-molekylerna är separerade på 15% denatureringen Polyakrylamidgelen (sida), där RNA oligonukleotiden storlek markörer som migrerats i intilliggande brun...

Watch this Scientific Journal Video about Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens at JoVE.com

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Formalin-fast paraffin-inbäddat exemplar representerar en värdefull källa av molekylära markörer av mänskliga sjukdomar. Här presenterar vi ett laboratoriebaserade cDNA bibliotek förberedelse protokoll, ursprungligen utformad med färsk fryst RNA och optimerad för analysen av arkiverade mikroRNA från vävnader lagras upp till 35 år.

Retrospektiv mikroRNA sekvensering: Kompletterande DNA bibliotek förberedelse protokoll använder Formalin-fast Paraffin-inbäddat RNA exemplar

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

The overall goal of this CDNA Library preparation is to enable successful sequencing of small RNAs that have been stored in formal and fixed paraffin-embedded tissues. This method is very useful for analyzing formal and fixed paraffin-embedded specimens. You extract RNA from up to 18 specimens, you analyze the content of all of these and obtain a profile of all of the microRNase for all the specimens.To begin the protocol, add 8.5 microliters of previously-prepared ligation master mix to each of the 18 individually aliquoted FFPERNA samples. Gently flick the tubes, and centrifuge for two seconds. After centrifuging, store the tubes on ice.Use a three microliter pipette to transfer one microliter of each adapter, to the corresponding FFPERNA samples containing RNA and ligation master mix. Do not pipette up and down, and change tips between FFPERNA samples. Close each tube, flick them to mix, and centrifuge them for two seconds.Set the tubes on ice. Denature the reactions by placing the tubes on a heat block at 90 degrees celsius for one minute, and then immediately place them on ice. Next, use a three microliter pipette to transfer one microliter of the diluted ligation enzyme into each of the 18 RNA samples.Set the ligations on ice, and place them in a four degrees celsius cold room for an overnight incubation of 18 hours. Place the tubes on a heat block for one minute at 90 degrees celsius to deactivate the ligation reaction and then return them to ice for at least two minutes to cool down. Transfer 1.2 microliters of the precipitation master mix into each tube.Add 63 microliters of 100%ethanol. Close each tube, flick the tubes to mix, and centrifuge them for two seconds. Place the tubes on ice.Combine the contents of all 18 tubes into a single 1.5 milliliter siliconized tube and invert three times to mix. Centrifuge the tubes for two seconds, and place them on ice for 60 minutes to precipitate. After precipitation, centrifuge the 1.5 milliliter tube with the pooled ligations.Next, remove the supernatant with a one milliliter pipette tip but leave some liquid at the bottom. Tilt the tube, and use a 20 microliter pipette to remove the remaining supernatant. Vacuum-suction the supernatant with a Pasteur pipette with a 10 microliter tip, then resuspend the RNA pellet in 20 microliters of RNase-free water by flicking the tube.Add 20 microliters of PAA gel loading solution to resuspend the RNA. Flick the tube, centrifuge the tube for two seconds at room temperature, and set the tube on ice. Replace the previously prepared 0.5 XTBE of the gel apparatus with fresh 0.5 XTBE, and load the latter, size markers, and ligated RNase in the center of the gel, leaving two empty wells on both sides.Then run the gel. After running the gel, uncast the 15%page by removing one of the glass plates. Lightly spray the gel sitting on the glass plate with 10 microliters of fluorescent dye solution and 25 milliliters of 0.5 XTBE, and let it sit flat for five minutes in the dark.Lay the glass with the gel on a blue-light transilluminator, and align both 19 nucleotide and 24 nucleotide size markers with a ruler to direct the excision of the ligated small RNAs. Then, excise the gel. Place the excised gel in a 0.5 milliliter tube designed to fragment gel slices, securely positioned into a 1.5 milliliter siliconized microcentrifuge tube.Centrifuge the gel at 16 thousand times G for three minutes at room temperature. Resuspend the fragmented gel with 300 microliters of 400 millimolar sodium chloride solution. Close the tube, and seal it with paraffin film.Agitate the contents on a thermo mixer at 11 hundred RPM in a four degrees celsius cold room for an overnight incubation. The next day, retrieve the tube from the thermo mixer and use a one milliliter pipette to transfer the solution onto a five micrometer filter tube, inserted into a 1.5 milliliter siliconized RNase-free collection tube. Centrifuge the tube for three minutes at 23 hundred times G at room temperature.Discard the filter tube, add 950 microliters of 100%ethanol to the collection tube containing the filtered solution, and invert the tube to mix. Centrifuge the tube for two seconds, and place it on ice for 60 minutes. Then, precipitate the RNA pellet.Next, dry the RNA pellet by opening the cap and carefully removing the supernatant with a one milliliter pipette, while leaving some liquid at the bottom. Use a 20 microliter pipette to remove all of the remaining supernatant without touching the pellet. Tilt the tube so any remaining solution moves to the opposite side of the pellet.Use a Pasteur pipette to aspirate the supernatant and dry the pellet using a vacuum. Resuspend the RNA pellet in 5.6 microliters of RNase-free water. Defrost the reverse transcription reagents on ice for 15 minutes.Set up a reverse transcription reaction by adding three microliters of 5X buffer, 4.2 microliters of 10X dioxynucleotide triphosphate, or DNTPs, and 1.5 microliter of dithiothreitol, or DTT. Gently flick the tube to mix the reaction, and centrifuge for two seconds to collect the solution. After the reaction is on a heat block at 90 degrees celsius for exactly 30 seconds, transfer it directly to a thermo mixer at 50 degrees celsius for two minutes to equalize the temperature.Add 0.75 microliters of the reverse transcription enzyme directly into the solution. Flick gently to mix, and set the tube back on the thermo mixer at 50 degrees celsius for 30 minutes immediately. After 30 minutes, transfer the tube to a heat block at 95 degrees celsius for one minute to stop the reverse transcription.Add 95 microliters of RNase-free water directly to the reverse transcription. Flick the tube to mix, and set it directly on ice for two minutes. Assemble the pilot PCR reaction and set up two PCR cycles on the thermo cycler.Set up the pilot PCR reaction on the thermo cycler and start file one. At the end of file one, use a 20 microliter pipette to transfer 12 microliters from the pilot PCR reaction into 1.5 milliliter microcentrifuge tube containing three microliters of 5X gel loading dye. Mix by pipetting up and down.And label it 10 cycles. Close the pilot PCR tube and start file two on the thermo cycler. At the end of file two, use a 20 microliter pipette to transfer 12 microliters of solution into a 1.5 milliliter microcentrifuge tube with three microliters of 5X gel loading dye, and label it 12 cycles.Repeat the steps above to collect 12 microliters of PCR products from the pilot PCR reaction at PCR cycles 14, 16, 18, and 20. Then add three microliters of 5X gel loading dye to each of the 12 microliter PCR aliquots, and to the 20 nucleotide ladder containing three microliters of ladder and nine microliters of RNase-free water. After running the samples on an agarose gel with 0.5 XTBE, evaluate the gel on a UV box to identify the optimal PCR cycle.Then set up the large-scale PCR reactions for agarose gel library purification. After PCR amplification, transfer nine microliters from each PCR tube into six 1.5 milliliter microcentrifuge tubes, containing three microliters of 5X gel loading dye. Prepare a 20 nucleotide ladder.Load the ladder, negative PCR control, and six PCR products onto the agarose gel, and run the gel for 30 minutes at 120 volts. Verify the uniformity of the PCR implifications between lanes on a UV box, and take an image. Combine three PCR reactions into two 1.5 milliliter siliconized microcentrifuge tubes, add 27 microliters of five molar sodium chloride, and 950 microliters of 100%ethanol.Invert the tubes to mix, and set them at 20 degrees celsius overnight to precipitate the PCR products. Using the protocol, the micro RNA expression correlation between the two fresh frozen tumor RNA and their specific FFPERNA specimen counterparts were ascertained. The plots between matched fresh-frozen and FFPE micro RNA indicate that the CDNA Library preparation provides a good reproducibility, as there is high correlation between the microRNA detected in specimens processed differently.To determine if the optimized CNDA Library preparation protocol was applicable and reproducible with increasingly older FFPE specimens, micro RNA expression profiles from FFPE tissues archived for 18, 20, 22, 27, 30, and 35 years were obtained. The correlation coefficient remained above 0.96 regardless of the age of the specimen or the library preparation week. Therefore, the optimized CDNA Library preparation protocol provides a robust tool for reproducible analysis of FFPE specimens, regardless of their archival time.So after watching this video you should have a good understanding on how to prepare your small RNA libraries and obtain a full microinexpression profile of all your precious formal and fixed paraffin-embedded specimens.

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