Name: absolute quantification of plasma microrna levels in cynomolgus monkeys, using quantitative real-time reverse transcription pcr
Uploaded: 2018-02-12T13:30:00
Description: Watch this Scientific Journal Video about Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR at JoVE.com

Questa ricerca non ha ricevuto alcuna sovvenzione specifiche da agenzie di finanziamento nel settore pubblico, commerciale, o non-profit.

Tutti gli esperimenti sono stati approvati dal istituzionale Animal Care e uso Comitato di Daiichi Sankyo Co., Ltd.
1. preparazione del campione
<ol><li>Raccogliere il sangue (almeno 0,5 mL) dalla vena femorale di scimmie cynomolgus in provette EDTA 2K-contenenti. Nota: Citrato o eparina non sono accettabili perché questi anticoagulanti inibiscono successive PCR14,15.</li>
	<li>Posizionare i campioni raccolti immediatamente sul g...

<ol>
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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=Absolute+Measurement+of+Cardiac+Injury-Induced+microRNAs+in+Biofluids+across+Multiple+Test+Sites.">Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.</a> Toxicol Sci. 154 (1), 115-125 (2016).</li><li>Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Analysis+of+Circulating+microRNA+Specific+to+the+Liver,+Heart,+and+Skeletal+Muscle+of+Cynomolgus+Monkeys.">Comprehensive Analysis of Circulating microRNA Specific to the Liver, Heart, and Skeletal Muscle of Cynomolgus Monkeys.</a> Int. J. Toxicol. 36 (3), 220-228 (2017).</li><li>Matsuzaka, Y., 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=Three+novel+serum+biomarkers,+miR-1,+miR-133a,+and+miR-206+for+Limb-girdle+muscular+dystrophy,+Facioscapulohumeral+muscular+dystrophy,+and+Becker+muscular+dystrophy.">Three novel serum biomarkers, miR-1, miR-133a, and miR-206 for Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, and Becker muscular dystrophy.</a> Environ. Health. Prev. Med. 19 (6), 452-458 (2014).</li><li>Starkey Lewis, P. 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A. 106 (11), 4402-4407 (2009).</li><li>Moldovan, L., Batte, K., Wang, Y., Wisler, J., Piper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+the+Circulating+MicroRNAs+in+Exosomes/Extracellular+Vesicles+from+Serum+or+Plasma+by+qRT-PCR.">Analyzing the Circulating MicroRNAs in Exosomes/Extracellular Vesicles from Serum or Plasma by qRT-PCR.</a> Methods Mol. Biol. 1024, 129-145 (2013).</li><li>Li, S., Chen, H., Song, J., Lee, C., Geng, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+heparin+inhibition+in+circulating+MicroRNAs+amplification.">Avoiding heparin inhibition in circulating MicroRNAs amplification.</a> Int. J. Cardiol. 207, 92-93 (2016).</li><li>Cheng, H. H., 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=Plasma+processing+conditions+substantially+influence+circulating+microRNA+biomarker+levels.">Plasma processing conditions substantially influence circulating microRNA biomarker levels.</a> PLoS One. 8 (6), e64795 (2013).</li><li>Serafin, 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=Identification+of+a+set+of+endogenous+reference+genes+for+miRNA+expression+studies+in+Parkinson's+disease+blood+samples.">Identification of a set of endogenous reference genes for miRNA expression studies in Parkinson's disease blood samples.</a> BMC Res. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+circulating+miRNAs+in+plasma:+effect+of+preanalytical+and+analytical+parameters+on+their+isolation+and+stability.">Quantification of circulating miRNAs in plasma: effect of preanalytical and analytical parameters on their isolation and stability.</a> J Mol Diagn. 15 (6), 827-834 (2013).</li><li>Yamaura, Y., Nakajima, M., Takagi, S., Fukami, T., Tsuneyama, K., Yokoi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+microRNA+profiles+in+rat+models+of+hepatocellular+injury,+cholestasis,+and+steatosis.">Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis.</a> PLoS One. 7 (2), e30250 (2012).</li><li>Kirschner, M. B., Edelman, J. J., Kao, S. C., Vallely, M. P., van Zandwijk, N., Reid, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Hemolysis+on+Cell-Free+microRNA+Biomarkers.">The Impact of Hemolysis on Cell-Free microRNA Biomarkers.</a> Front Genet. 4, 94 (2013).</li><li>Mestdagh, P., 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=High-throughput+stem-loop+RT-qPCR+miRNA+expression+profiling+using+minute+amounts+of+input+RNA.">High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.</a> Nucleic Acids Res. 36 (21), e143 (2008).</li><li>Pritchard, C. C., Cheng, H. H., Tewari, 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+profiling:+approaches+and+considerations.">MicroRNA profiling: approaches and considerations.</a> Nat. Rev. Genet. 13 (5), 358-369 (2012).</li><li>Moldovan, L., Batte, K. E., Trgovcich, J., Wisler, J., Marsh, C. B., Piper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+challenges+in+utilizing+miRNAs+as+circulating+biomarkers.">Methodological challenges in utilizing miRNAs as circulating biomarkers.</a> J. Cell. Mol. 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La nostra valutazione globale fornito un'analisi statistica più rigorosa dell'estensione della gamma dinamica, che ha chiaramente indicato che la grandezza della variazione tra singoli campioni era estremamente diversa tra i miRNA testati. Anche se queste variazioni possono essere attribuibili ai loro piccoli quantitativi nei liquidi corporei, si deve osservare che questi dati riflettono non solo variazioni biologiche, ma anche varianti tecniche. La maggior parte della variazione tecnica può essere valutata mediante va...

Un numero crescente di studi è stati esplorare i microRNA (Mirna) come biomarcatori per la diagnosi e la prognosi di tumori, o di monitoraggio e rilevamento di altre malattie in studi non clinici e clinici1,2,3 . Trascrizione d'inversione quantitativa Real-Time PCR (RT-qPCR) è uno dei metodi più comuni utilizzati per valutare i miRNAs obiettivo individuale, perché questa tecnica è più sensibile la sequenza di RNA e microarray4 basato su piattaforme5. In generale, espressione dei miRNA è misurata rispetto a un campione di riferimento utilizzando il metodo di ΔCq6. Questo approccio è adatto per indagare i cambiamenti fisiologici nei livelli di espressione del gene bersaglio. Tuttavia, quantificazione relativa di Mirna in circolazione ha limitato l'utilità a causa delle loro piccole quantità. Inoltre, variazione tecnica rende difficile confrontare i risultati di diversi studi, perché diversi laboratori personalizzare i protocolli sperimentali di RT-qPCR in modo diverso, che porta a incoerente o contraddittoria anche risultati da diversi studi7.
In considerazione delle problematiche di cui sopra, quantificazione assoluta potrebbe essere più adatto per la valutazione delle quantità piccole di miRNA nei liquidi corporei. Il metodo di quantificazione assoluta utilizza una curva standard generata da concentrazioni note di oligonucleotidi sintetici di RNA che sono identici in sequenza per la corrispondente destinazione miRNA8. La salute e l'Istituto di scienze ambientali (HESI) Comitato tecnico sulla genomica ha recentemente condotto studi completi per confrontare i risultati di misure assolute di Mirna al plasma, in più siti di prova. I risultati hanno mostrato che utilizzando un protocollo standard per la quantificazione assoluta di Mirna ha dato risultati comparabili tra i siti di più prova9. Il metodo di analisi di RT-qPCR descritto nel presente studio è quasi identico al protocollo standard di HESI, che include analisi "multiplex" di molteplici obiettivi di miRNA e pre-amplificazione per facilitare il rilevamento di bassa espressione di Mirna.
In questo studio, un volume fisso (200 µ l) di plasma EDTA preparato dal sangue raccolti dalla vena femorale di scimmie cynomolgus cosciente (n = 50) è stato usato10. Il seguente protocollo descrive la procedura per la preparazione di campioni di plasma, estrazione di miRNA e RT-qPCR, tra cui pre-amplificazione. Ancora più importante, ulteriori informazioni tecniche sul protocollo sono stato inclusi, affinché la quantità di destinazione miRNA nei campioni possa essere convalidata in combinazione con un processo ben qualificato. In primo luogo, la curva standard di ciascun miRNA è stata validata per la sua gamma di rilevazione individuale, prima della relativa quantificazione in campioni biologici. In secondo luogo, la qualità della metodologia corrente completamente è stata valutata per mezzo di valori Cq di un controllo esterno (cel-miR-238). Pertanto, questa piattaforma produce dati più informativi e affidabili per confrontare i risultati di diversi studi o laboratori.
I profili dei 8 miRNA sono stati inclusi in questo rapporto come risultati rappresentativi dal metodo di analisi descritto qui. Questi miRNA sono stati proposti come potenziali biomarcatori di sicurezza associate a lesioni dei tessuti al fegato (miR-122 e miR-192), cuore (miR-1, miR-208a, miR-208b e miR-499a) e muscolo scheletrico (miR-133a e miR-206) in roditori ed esseri umani3, 11,12,13.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">BD Microtainer tube (K2EDTA)</td>
			<td style="width:195px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">365974</td>
			<td style="width:167px;">For blood collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf PCR Tubes, 0.2 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">0030124359</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Safe-Lock micro test tubes&#160; 1.5 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">0030120086</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Safe-Lock micro test tubes&#160; 2.0 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">0030120094</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Synthetic oligonucleotide</td>
			<td style="width:195px;">Hokkaido System Science</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Individual miRNA (0.2 &#956;mol,HPLC grade)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris-EDTA Buffer&#160; (pH 8.0)</td>
			<td style="width:195px;">Nippon Gene</td>
			<td style="width:119px;">314-90021</td>
			<td style="width:167px;">TE buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Buffer RPE</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Contents in miRNeasy mini kit&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Buffer RWT</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Contents in miRNeasy mini kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miRNeasy Mini Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">217004</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease-Free Water&#160;</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">129114</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAzol Lysis Reagent</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Contents in miRNeasy mini kit&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syn-cel-miR-238-3p miScript miRNA Mimic&#160;</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">219600</td>
			<td style="width:167px;">ID:MSY0000293, 5 nmol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SC Adapters</td>
			<td style="width:195px;">TAIGEN Bioscience Corporation</td>
			<td style="width:119px;">S0120</td>
			<td style="width:167px;">For RNA extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VacEZor 36 Complete System</td>
			<td style="width:195px;">TAIGEN Bioscience Corporation</td>
			<td style="width:119px;">M3610</td>
			<td style="width:167px;">For RNA extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">7900HT Fast Real-Time PCR System</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4351405</td>
			<td style="width:167px;">Fast 96-Well Block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneAmp PCR System 9700</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">9700</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MicroAmp Fast Optical 96-Well Reaction Plate</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4346907</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MicroAmp Optical Adhesive Film</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4311971</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Fast Advanced Master Mix</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4444557</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (cel-miR-238-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000248</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-122-5p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002245</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-133a-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002246</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-1-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002222</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-192-5p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000491</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-206)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000510</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-208a-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000511</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-208b-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002290</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-499a-5p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 001352</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Reverse 
			Transcription Kit</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4366597</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan PreAmp Master Mix (2&#215;)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4391128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform&#160;</td>
			<td style="width:195px;">Wako Pure Chemicals</td>
			<td style="width:119px;">035-02616</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol (99.5)</td>
			<td style="width:195px;">Wako Pure Chemicals</td>
			<td style="width:119px;">057-00456</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

absolute quantification of plasma microrna levels in cynomolgus monkeys, using quantitative real-time reverse transcription pcr

RT-qPCR è uno dei metodi più comuni per valutare i miRNA obiettivo individuale. Livelli di Mirna sono generalmente misurati rispetto a un campione di riferimento. Questo approccio è appropriato per l'esame di cambiamenti fisiologici nei livelli di espressione del gene bersaglio. Tuttavia, la quantificazione assoluta usando meglio l'analisi statistica è preferibile per una valutazione completa dei livelli di espressione genica. Quantificazione assoluta è ancora non di uso comune. Questo rapporto descrive un protocollo per misurare i livelli assoluti di miRNA al plasma, usando RT-qPCR con o senza pre-amplificazione.
Un volume fisso (200 µ l) di plasma EDTA è stato preparato dal sangue raccolto dalla vena femorale di scimmie cynomolgus cosciente (n = 50). il RNA totale è stato estratto utilizzando sistema commercialmente disponibile. Al plasma miRNA sono stati quantificati dall'analisi di RT-qPCR sonda-based che contiene specifici miRNA avanti/indietro PCR primer e sonda. Le curve standard per quantificazione assoluta sono state generate utilizzando oligonucleotidi da RNA sintetici disponibile in commercio. Un sintetico cel-miR-238 è stato usato come un controllo esterno per la valutazione di qualità e di normalizzazione. I miRNA che ha mostrato la quantificazione ciclo (Cq) valori superiori a 35 sono stati pre-amplificati prima il passo di qPCR.
Tra i 8 Mirna esaminati, miR-122, miR-133a e miR-192 erano rilevabili senza pre-amplificazione, considerando che miR-1, miR-206 e miR-499a necessari pre-amplificazione a causa dei loro livelli di espressione bassa. MiR-208a e miR-208b non erano rilevabili anche dopo pre-amplificazione. L'efficienza di elaborazione del campione è stata valutata dai valori Cq di spillo cel-miR-238. In questo metodo di analisi, tecnica variazione è stata stimata in meno di 3 volte e il limite inferiore di quantificazione (LLOQ) era 102 copia / µ l, per la maggior parte dei miRNA esaminati.
Questo protocollo fornisce una migliore stima della quantità di plasma Mirna e permette la valutazione della qualità dei dati corrispondenti da diversi studi. Considerando che il basso numero di miRNA nei liquidi corporei, pre-amplificazione è utile per migliorare la rilevazione di mal espresso Mirna.

Flusso di lavoro di analisi di miRNA da RT-qPCR e la valutazione di qualità deit La figura 1 Mostra il flusso di lavoro di analisi di miRNA da campioni di sangue usando qPCR10. La qualità degli esperimenti può essere verificata includendo cel-miR-238 come un controllo esterno. Questo rivelerà varianti tecniche in estrazione del RNA e i processi successivi RT-qPCR. In questo studio, la media ± SD di Cq ...

Watch this Scientific Journal Video about Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR at JoVE.com

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

Questo rapporto descrive un protocollo per misurare i livelli assoluti di miRNA al plasma, usando trascrizione d'inversione quantitativa Real-Time PCR, con o senza pre-amplificazione. Questo protocollo offre una migliore comprensione della quantità di plasma Mirna e permette la valutazione qualitativa dei dati corrispondenti da diversi studi o laboratori.

Quantificazione assoluta dei livelli di MicroRNA del Plasma nella scimmia cynomolgus, mediante trascrizione d'inversione quantitativa Real-Time PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

The overall goal of this procedure is to measure the absolute levels of Plasma MicroRNAs using Quantitative Real-time Reverse Transcription PCR. This method can help assess the quantity of plasma microRNAs, even if their expression level is low. The main advantage of this technique is that the measured data can be compared with others from different studies or laboratories.The implications of this technique extend to the samples from other species because a standard curve can be generated by using the synthetic RNA or the nucleotide. This method can provide insight into translational research to investigate promising safety biomarkers. First, thaw the frozen plasma samples obtained from the femoral vein of the Cynomolgus Monkeys on ice.Transfer the Lysis reagent in Chloroform on ice for chilling before RNA extraction. Next, add 1, 000 microliters of Lysis reagent to 200 microliters of the sample. And vortex for one minute to ensure proper mixing.Then add five microliters of five nanomolar synthetic Caenorhabditis elegans microRNA and 200 microliters of chloroform to the sample. Vortex the sample for one minute to mix properly. Then, place the sample on ice for two to three minutes.Next, centrifuge the sample at 12, 000 G, for 15 minutes at four degrees Celsius. Next, carefully transfer 650 microliters of the aqueous phase to a new microtube. Then, add 975 microliters of Ethanol to the aqueous phase and pipette several times to ensure complete mixing.Transfer the sample into the corresponding column and adapter and then vacuum dry for three minutes using vacuum manifolds. Then add 200 microliters of Ethanol to the column and vacuum dry for one minute. After vacuum drying, add 800 microliters of RWT Buffer to the column and, again, vacuum dry for two minutes.Then add 800 microliters of RPE Buffer to the column and vacuum dry for two minutes. Add the RPE Buffer twice, followed by vacuum drying. Next, add 300 microliters of Ethanol to the column.After adding Ethanol, vacuum dry for one minute. Then, transfer the column in a new microtube and centrifuge the same at 12, 000 G at room temperature for one minute. After centrifugation, transfer the column to a new microtube and add 50 microliters of Nuclease-free water to it.Let the column stand at room temperature for three minutes and then centrifuge at 8, 000 G at room temperature for one minute. Finally, store the eluate at minus 80 degrees Celsius until use. To prepare a synthetic RNA Oligonucleotide Concentration that corresponds to the target microRNA, dilute the stock solution 10-fold to achieve a working solution with the highest concentration to plot the standard curves.Next, to constitute a multiplex pool of reverse transcription primers, mix equal volumes of the 20-strength primers for target microRNAs. Then, prepare the reverse transcription reaction mix. Then add 10 microliters of reverse transcription reaction mix to five microliters of RNA sample.Mix the two by pipetting several times. Then, incubate the mixture on ice for five minutes. After placing the sample in the Thermocycler, start the cycle.Once the program is over, store the reverse transcribed sample at minus 80 degrees Celsius. To constitute the multiplex assay primer pool, add equal volumes of five microliters of assay primers corresponding to target microRNAs into the tube containing Tris-EDTA buffer in the final volume of 1, 000 microliters. Next, constitute the pre-amplification reaction mix.Once the reaction mix is done, transfer 22.5 microliters of pre-amplification reaction mix to two point five microliters of reversed-transcribed sample. Then, pipette to mix the sample and pre-amplification master mix thoroughly and incubate on ice for five minutes and then leave the PCR tubes in the Thermocycler and start the run. Once the program is over, transfer all the samples at minus 80 degrees Celsius.After thawing the pre-amplified or non-pre-amplified samples, dilute it five-folds with sterile water. To generate the standard curve, do 10-fold serial dilution of the samples derived from synthetic RNA oligonucleotides. Then, constitute the quantitative PCR reaction mix in a tube kept on ice.Then add 18 microliters from the quantitative PCR reaction mix to the Fast Optical 96-Well Reaction Plates. Then add two microliters of the diluted samples into the wells. After sealing the plate with adhesive film, briefly centrifuge the sample at 500 G for 15 seconds.Then start the real-time Thermocycler program. Use a SDS software Version 2.4 that functions with the corresponding real-time thermal cycler to compute the raw copy number of each sample. Then calculate the absolute copy number from the raw copy number using Excel software.Amplification efficiency of miR-122, miR-192, and miR-133a was analyzed by plotting standard curves, which explains the relation between the log concentration versus the quantification cycle for the non-pre-amplified samples. The standard curves for miR-122, miR-192, and miR-133a show a linear relationship between quantification cycles and log concentration of the samples. Next, amplification efficiency of miR-1, miR-499a, and miR-206 was analyzed by plotting standard curves for pre-amplified samples.To analyze the amplification efficiency, the slope was computed using linear regression. The slope in standard curve A shows the absence of specific amplicons at 1, 000 copies per microliter concentration for miR-499a and miR-206. However, the non-specific amplicons were obtained at 1, 000 copies per microliter for miR-1.Here, non-pre-amplified and pre-amplified miR-206 samples were used in duplicates to derive the amplification plots. The quantification cycle values for non-pre-amplified and pre-amplified miR-206 was estimated to be 39.9, plus or minus one point nine and 27.0, plus or minus two point two, respectively, representing almost similar values between the duplicates as shown in the plots. Next, plasma microRNAs obtained from Cynomolgus monkeys were subjected to profiling to compute their absolute values.The dot plots, obtained from microRNA profiling representing their expression levels, shows that miR-122, miR-133a, and miR-192 is detectable without pre-amplification. Whereas, miR-1, miR-206, and miR-499a will require pre-amplification due to low expression levels. After watching this video, you should have a good understanding of how to measure the absolute levels of plasma microRNA, using RT-qPCR.And pre-amplification;it's useful to improve detection with small quantities of microRNAs.

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