저자 아무 승인 있다.

1. 혈장/혈 청에서 miRNA의 추출
참고: 계량 miRNAs 적절 하 게, 위하여 혈장/혈 청 으로부터 분리 하 여 정확한 예측에 관한 중요 한 단계입니다. 명심할 것, 특히 때문에 다른 프로토콜, 중요 한 건 샘플을 처리 하는 동안 동일한 워크플로를 준수 하는 것입니다. 이 프로토콜에서 miRNA는 혈 청의 50 µ L에서 추출 됩니다. 이 제한은 올바른 추출 과정으로 200 µ L를 사용 하지 마십시오.
<ol><li>혈 청을 준비 하거나 냉동된 샘플을 동결 해제 합니다.</li>
	<li>혈 청의 50 µ L를 250 µ L (5 권) 상업 세포 시 약을 추가 합니다. Vortexing 여는 세포를 지원 합니다. 5 분 동안 실 온에서 벤치에는 lysate와 튜브를 놓습니다.</li>
	<li>스파이크에 컨트롤 (107 복사본 / µ L x 1.6)의 3.5 µ L로 lysate 스파이크와 vortexing에 의해 그들을 섞는다.</li>
	<li>단계 구분에 대 한 클로 프롬 (즉, 시작 혈 청 샘플으로 동일한 금액)의 50 µ L를 추가 합니다. 15 미 위 2-3 분 동안 실 온에서 벤치에 튜브 튜브를 적극적으로 악수.</li>
	<li>단계 구분에 대 일 분 동안 4 ° C에서 12000 x g에서 튜브 원심. 참고: 샘플 이제 RNA, 흰색 interphase, 그리고 낮은, 핑크 유기 단계를 포함 하는 위 수성 단계로 구분 됩니다.</li>
	<li>위 수성 단계 (100 µ L) RNA를 포함 하는 새로운 관으로 이동. 이 올바른 RNA 수 위조로 흰색 interphase 자료를 전송 하지 않습니다.</li>
	<li>µ 150 L (즉, 전송된 샘플의 볼륨 x 1.5) 100% 에탄올을 추가 합니다. 그들을 아래로 pipetting으로 재료를 믹스. 그들, 원심 고 신속 하 게 다음 단계로 이동 하지.</li>
	<li>2 mL 컬렉션 튜브에서 상업 회전 열에 샘플의 250 µ L 플라스틱 뚜껑을 닫습니다. 원심에서 열 > 흐름 통해 15 미 삭제에 대 한 실 온에서 8000 x g.</li>
	<li>스핀 열에 상업적인 세척 버퍼 번호 1의 700 µ L를 추가 합니다. 뚜껑을 닫고 원심에서 열 > 8000 x g 15 미 삭제 실행을 통해 세척 버퍼에 대 한 실내 온도에.</li>
	<li>스핀 열에 상업적인 세척 버퍼 번호 2의 500 µ L를 추가 합니다. 뚜껑을 닫고 원심에서 열 > 8, 000 15 미 삭제 실행을 통해 세척 버퍼에 대 한 실 온에서 g x.</li>
	<li>스핀 열 80% 에탄올 500 µ L 플라스틱 뚜껑을 닫고, 원심에서 열 > 8, 000 x g 2 분에 대 한 실 온에서 흐름 통해와 수집 튜브 삭제.</li>
	<li>새로운 2 mL 컬렉션 튜브를 회전 열을 전송 합니다. 열 건조는 막 열린된 뚜껑 최고 속도로 원심 플로우 스루와 수집 튜브를 삭제 합니다.</li>
	<li>새로운 1.5 mL 컬렉션 튜브로 스핀 열을 전송 합니다. 막의 센터에 직접 RNase 무료 물 30 µ L을 적용 하 여 RNA을 elute. 뚜껑을 닫습니다. 1 분에 대 한 최고 속도로 열 원심</li>
	<li>-70 ° c.에 RNA를 저장 참고: RNase 무료 물-70 ° C ~-20 ° C 사이에서 순화 된 RNA의 저장 하면 1 년에 대 한 RNA의 저하 없이. 제조 업체의 프로토콜에 따라 RNase 무료 물 희석 RNA의 최소 금액은입니다 10 µ L. 10 µ L 수 있습니다 충분히 수 화 막 그리고 총 RNA 수확량 감소를 사용 하 여.</li>
</ol>2. 반전 녹음 방송
참고: 보완 DNA (cDNA) 다음 15 µ L 반전 녹음 방송 (RT) 프로토콜 (총 반응 볼륨)를 사용 하 여 합성 되었다.
<ol><li>RT 키트 얼음에 녹여 냉장고 만큼 저하에서 그들을 방지 하 고 사용 하기 전에 그들을 아래로 회전 수에 효소를 유지. 얼음에 RT 뇌관을 해 동 하 고 사용 하기 전에 그들을 아래로 회전.</li>
	<li>표 1에 설명 된 대로 마스터 믹스를 준비 합니다.</li>
	<li>믹스 마스터의 10 µ L 당 96 잘 접시에 잘 추출 된 RNA의 5 µ L을 결합.</li>
	<li>2 분 동안 4 ° C에서 2000 x g에 잘 접시 원심</li>
	<li>반전 녹음 방송에 대 한 표 2 에 설명 된 열 주기 프로토콜을 사용 합니다. 참고: 아래 설명 된 대로 cDNA 직접 처리 될 수 있습니다 디지털 PCR 기계에 ( 재료의 표참조). 또는, 그것은-20 ° c.에 저장 될 수 있습니다.</li>
</ol>3. 물방울 생성 및 디지털 PCR
참고: 디지털 PCR 다음 40 µ L 프로토콜을 사용 하 여 수행 되었다.
<ol><li>해 동 상업 dPCR 믹스, 준비 된 cDNA 및 얼음에 PCR 뇌관. 사용 하기 전에 잠시 그들을 원심.</li>
	<li>에 설명 된 대로 마스터 믹스를 준비 표 3.
</li>
	<li>RT 제품의 1.33 μ/잘 추가 하 고 간단히 원심.</li>
	<li>8 잘 카세트 (중간 행)의 각 음에 샘플의 20 μ를 플라스틱. 참고: 8 잘 카세트 완전 하 게 채워지지 경우 채울 cDNA (즉, 추출 된 RNA 대신 물으로 생산 하는 실시간 제품)을 생략 하는 템플릿이 아닌 컨트롤 (NTCs)와 나머지 우물 또는 dPCR 상업 버퍼. NTC는 오염 제어 역할을 합니다. 물 그 방울 형성의 품질에 악영향을 줄 것입니다 때문에 나머지 우물에서 사용할 수 없습니다.</li>
	<li>8 잘 카세트의 유 정 (아래쪽 행)으로 프로브에 대 한 작은 물방울 생성 기름의 70 μ를 플라스틱.</li>
	<li>8 잘 카세트 위에 가스 켓을 놓고 방울 생성기에서 카세트를 배치 합니다. 드롭릿 생성기를 닫습니다. 3 표시등은 녹색으로 고정 될 때까지 기다립니다. 참고: 방울 지금 생성 되고있다.</li>
	<li>신중 하 고 천천히 pipetting으로 96 잘 접시의 별도 우물으로 물방울 형성 샘플 (맨 윗줄)의 40 μ를 전송.</li>
	<li>샘플에 작은 물방울 대형을 완료 한 후 인감 4 180 ° C에서 pierceable dPCR 호 PCR 플레이트 s.</li>
	<li>호 봉인 PCR-접시는 cycler 놓고 2.5의 경사로 속도로 표 4 에 설명 된 대로 주기 ° C/s.</li>
</ol>4. 물방울 읽기 및 분석
<ol><li>플레이트 홀더 베이스에 열 cycler에서 접시를 전송. 상단의 PCR 플레이트에 플레이트 홀더를 배치 하 여 PCR 접시를 조입니다.</li>
	<li>상업적인 dPCR 소프트웨어를 시작 합니다.</li>
	<li>설정에서 샘플 이름, 실험, 그리고 대상의 이름을 입력 합니다. 절대 정량화를 선택 합니다.</li>
	<li>실행을 클릭 하 여 읽기 방울을 시작 합니다. 가수분해 프로브를 작업할 때 탐지 화학으로 팸/HEX 또는 팸/빅 을 선택 합니다. 참고: 상업 dPCR 소프트웨어 다음 자동-분석 데이터.</li>
	<li>올바른 물방울 생성 되도록 결과 테이블에 물방울 번호를 확인 하십시오.</li>
	<li>같은 정량된 miRNA와 모든 우물을 선택 하 고 분석을 클릭 합니다. 2D 오를 사용 하 여 긍정적인 물방울 표시 하 고 수동으로 수정 자동 분석 데이터 (그림 1).</li>
</ol>5. 올바른 샘플 계산
<ol><li>샘플 샘플 정규화 인자 (NF)를 곱하여 정규화. NF = 중간 [C. 선 충 미르-39 측정]모든 샘플/ [선 충 C. 미르-39]주어진된 샘플
</li>
	<li>마지막 미르 혈 청 농도, 희석 요인 (DF)에 대 한 조정 계산 합니다. [미르] 마지막 = [미르]원시 값 DFRT DFdPCR DFEx x x x 장소: [미르] 원시 값 = 상업 dPCR 소프트웨어에 의해 계량 미르 DFRT 반전 녹음 방송;에 서식 파일의 희석 비율 = DFdPCR = dPCR;에 서식 파일의 희석 비율 Ex DF = 희석 요인 추출에서.</li>
</ol>6. 합성 Oligonucleotide 희석 시리즈
<ol><li>동결 건조 된 합성 oligonucleotide 얼음에 녹여 짧게 원심 그것.</li>
	<li>동결 건조 된 합성 oligonucleotide nuclease 무료 물 10 pmol의 최종 농도에 희석 / µ L. 간단히 원심 그것.</li>
	<li>표 5에 설명 된 대로 nuclease 무료 물에 희석. 8000 x g 10에 각 희석 단계 사이 짧게 희석 원심 s.</li>
	<li>2 단계에서 설명한 대로 반전 녹음 방송으로 계속 하 고 3 단계와 4 단계에서 설명한 대로 디지털 PCR와 샘플을 분석.</li>
</ol>

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저자는 공개 없다.

디지털 PCR PCR는 샘플 내에서 핵 산의 직접 절대 정량화를 허용 하는 상대적으로 새로운 끝점 방법입니다. 메서드를 사용 하면 감소 변화, 증가 일상적인 재현성 및 우수한 감도11,12를 포함 하 여 특정 장점을 있습니다. 또한, 분할 샘플의 약 20000 단일 반응 및 끝점 분석으로, 때문에 dPCR는에 더 강력한 PCR 양적 RT-PCR16에 비해 간섭 물질. DP...

순환 miRNAs 질병, 심혈 관 질환1등의 숫자에 대 한 유망 표식으로 확인 되었습니다. miRNAs는 작은, 비 코딩 단일 가닥 RNA 분자 (약 22 뉴클레오티드 긴) 관련은 post-transcriptional 레 귤 레이 션 을 통해 메신저 RNA 번역 및 영향을 미치는 유전자 식2, 변경에 생리 적 및 병 적인 상태에 순환으로 해제 됩니다. 특정 miRNAs의 양적 표현, 병 리에 연결 될 수 있습니다 그리고 일부 miRNAs 보여 높은 조직과 질병 특이성1. 심혈 관 질환, miRNAs 되고있다 매력적인 후보자로 소설 생체 PCR 방법론3의 도움으로 측정할 수 있기 때문에 그들은 혈 청에 있는 현저 하 게 안정 하 고 쉽게 할 수 있다. 작은 연구에서 심근 경색에 대 한 생체로 miRNAs의 잠재적인 가치를 평가 하지만 큰 동료에서 유효성 검사2부족 이다. 예를 들어 미르-499 이다 높은 심근 근육 표현 발견 하 고 아미4,,56에 크게 증가를 보였다. 그것은 또한, 조절 프로그램 된 세포 죽음 (apoptosis)와 cardiomyocytes의 차별화 및 따라서 AMI7다음 몇 가지 메커니즘에 관련 된. 우월과 아미의 진단에 대 한 miRNAs의 증분 값을 보고 몇 가지 작은 연구를 제외 하 고 우월 또는 높은 감도 심장 troponins 평등 입증 되지 않은 아직 대규모 연구2,5 ,,68. 큰 동료에서 더 많은 예비 연구는, 따라서, miRNAs의 잠재적인 진단 가치를 평가할 필요 합니다. 또한, miRNA 정량화의 방법을 최적화 하 고9프로토콜 표준화를 사용 하 여 비교. 표준화 된 분석 실험 일관성 없는 결과 줄일 수 있습니다 하 고 일상적인 임상 응용 프로그램에 대 한 잠재적인 생체 되 miRNAs biomarkers 그들의 임상 적용 되도록 재현 방법으로 양이 정해질 필요가 도움이 될 수 있습니다.
최근, dPCR 끝점 분석으로 도입 되었습니다. 그것은 약 20000 개별 반응10샘플 파티션을. DPCR 시스템은 다음 수학 포아송 통계 분석 형광 신호 (긍정적이 고 부정적인 반응)의 표준 곡선10없이 절대 정량화를 사용를 사용 합니다. 때 dPCR를 결합 하 여 형광 가수분해 프로브, miRNAs의 매우 구체적인 직접 절대 정량화 가능한 이루어집니다. 디지털 연쇄 반응으로 나타났습니다 미르 수준 측정에 대 한 (를 포함 하 여 감소 변화, 증가 일상적인 재현성, 선형성의 고차 및 높은 감도) 우량한 기술적인 질을 전시 하는 순환 양적 실시간 PCR10,11에 비해. 이러한 우수한 기술적 특성 생체로 순환 miRNAs를 사용 하 여 현재 한계를 완화 하는 데 도움이 될 수 있습니다. 및 biomarkers 대형 멀티 센터 심장 혈관 임상에서과에서 진단 방법으로 miRNAs의 설립으로 이어질 수 있습니다. 일반. 이전 연구에서 우리는 최근 miRNAs는 AMI 환자의 순환의 절대 정량화에 대 한 dPCR를 적용 하 고 정량12miRNAs의 정량화에 비해 우수한 진단 가능성을 입증 할 수 있었다.
이 출판물에는 dPCR를 사용 하 여 직접 심장 혈관 miRNAs 순환 측정에 대 한 정확 하 고 재현 방법 설명 하겠습니다. 혈 청, 디지털 PCR를 사용 하 여 미르 수준 절대 정량화 대형 멀티 센터 심장 혈관 임상 시험에 사용에 대 한 잠재적인 보여줍니다. 이 책에서 우리가 자세히 설명 효과적으로 디지털 PCR을 수행 하는 방법 및 혈 청에 절대 미르 복사본 수를 검출 하는 방법.

<table>
	<tbody>
		<tr>
			<td>RNA-Extraction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Kit (50)</td>
			<td>Qiagen-Sample &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>217184</td>
			<td>Kit for microRNA extraction. Kit contains commercial buffer RWT (called number one in the manuscript)&#160; and RPE (called number two in manuscript).</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in-Control; Syn-cel-miR-39 miRNA; 10pmol</td>
			<td>Qiagen-Sample &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>219610</td>
			<td>Spike-in for normalisation , Sequence: 5&#39;-UCACCGGGUGUAAAUCAGCUUG-3&#39;</td>
		</tr>
		<tr>
			<td>Reverse Transcription</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit (1000 Reactions)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4366597</td>
			<td>Kit for microRNA reverse transcription</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Assays M</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assays used in reverse transcription</td>
		</tr>
		<tr>
			<td>hsa-miR-499 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 001352</td>
		</tr>
		<tr>
			<td>cel-miR-39 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 000200</td>
		</tr>
		<tr>
			<td>PCR Plate, 96-well, segmented, semi-skirted</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>AB0900</td>
			<td>96 well plate for reverse transcription</td>
		</tr>
		<tr>
			<td>Microseal &#8216;B&#8217; seal Seals</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>MSB1001</td>
			<td>Foil to ensure proper storage</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1851196</td>
			<td>Cycler used for reverse transcription</td>
		</tr>
		<tr>
			<td>Droplet Digital PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 nmole RNA oligo hsa-miR-499-5p</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>Sequence: 5&#39;-phos-UUAAGACUUGCAGUGAUGUUU-3&#39;</td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP)</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863024</td>
			<td>Supermix used in droplet generation</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Assays</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assays used in digital PCR (fluorescent hydrolysis probe)</td>
		</tr>
		<tr>
			<td>hsa-miR-499 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 001352, commercial primers</td>
		</tr>
		<tr>
			<td>cel-miR-39 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 000200, commercial primers</td>
		</tr>
		<tr>
			<td>DG8 Cartridges and Gaskets</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1864007</td>
			<td>Cartridge takes up to 8 samples for droplet generation</td>
		</tr>
		<tr>
			<td>DG8 Cartridge Holder</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863051</td>
			<td>Holds cartridges in droplet generation</td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863005</td>
			<td>Oil used in droplet generation</td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>12001925</td>
			<td>96 well plate for ddPCR</td>
		</tr>
		<tr>
			<td>PCR Plate Heat Seal, foil, pierceable</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1814040</td>
			<td>Pierceable foil, compatible with droplet reader</td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863004</td>
			<td>Oil used in droplet reading</td>
		</tr>
		<tr>
			<td>QX100 or QX200 Droplet Generator</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863002</td>
			<td>Droplet Generator, generates the droplets from sample/oil emulsion</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1814000</td>
			<td>Seals the plate before PCR</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1851196</td>
			<td>Cycler used for ddPCR</td>
		</tr>
		<tr>
			<td>QX100 or QX200 Droplet Reader</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863003</td>
			<td>Reads PCR-positive and PCR-negative droplets with an optical detector</td>
		</tr>
		<tr>
			<td>ddPCR Buffer Control for Probes</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863052</td>
			<td>Blank control&#160;and to fill up the remaining wells of 8-well cassette</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantaSof Software</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1864011</td>
			<td>Program for droplet reading</td>
		</tr>
		<tr>
			<td>Prism Windows 5</td>
			<td>GraphPad Software Inc., La Jolla, CA, USA</td>
			<td></td>
			<td>Program for statistical analysis</td>
		</tr>
	</tbody>
</table>

digital pcr for quantifying circulating micrornas in acute myocardial infarction and cardiovascular disease

순환 혈 청 microRNAs (miRNAs) 순환으로 심장 혈관 세포에서 출시 되는 심혈 관 질환 및 급성 심근 경색 (AMI), 대 한 생체로 약속을 보여왔다. 순환 miRNAs 매우 안정 하 고 측정할 수 있습니다. 특정 한 miRNAs의 양적 표현 병 리, 그리고 일부 miRNAs 쇼 높은 조직 및 질병 특이성에 연결할 수 있습니다. 심혈 관 질환에 대 한 새로운 바이오 마커를 찾는 의료 연구에 대 한 중요성 이다. 아주 최근에, 디지털 연쇄 반응 (dPCR) 발명 되었습니다. dPCR, 형광 가수분해 프로브와 함께 특정 직접 절대 정량화를 수 있습니다. dPCR 낮은 다양성, 높은 선형성 및 정량적 중 합 효소 연쇄 반응 (정량)에 비해 높은 감도 포함 하 여 우수한 기술적 특성을 전시 한다. 따라서, dPCR miRNAs, 특히 대형 멀티 센터 심장 혈관 임상에서 사용에 대 한 직접 측정 하는 더 정확 하 고 재현 가능한 방법 이다. 이 책에서 우리는 효과적으로 혈 청 샘플에 절대 복사 번호 평가 디지털 PCR을 수행 하는 방법을 설명 합니다.

디지털 PCR 형광 가수분해 프로브와 함께 연구를를 직접 복사본 / µ L에서 특정 miRNAs의 절대 금액을 정할 수 있습니다. DPCR에서 샘플 dPCR 기술 하지 않아도 약 20000 개별 PCR 반응에서 분할로10을 복제 합니다. DPCR 시스템 활용 통계 분석 형광 신호 (긍정적이 고 부정적인 반응 사이 다른)의 푸아송의 수학 곡선10의 표준에 대 한 필요 없이 절?...

Watch this Scientific Journal Video about Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease at JoVE.com

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

순환 microRNAs는 심혈 관 질환 및 급성 심근 경색에 대 한 생체로 약속을 보여주었다. 이 연구에서 우리는 미르 추출, 반전 녹음 방송 및 심혈 관 질환 환자의 혈 청에서 miRNAs의 절대 정량화에 대 한 디지털 PCR 프로토콜을 설명합니다.

급성 심근 경색 및 심혈 관 질환 MicroRNAs 순환 측정을 위한 디지털 PCR

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

digital-pcr-for-quantifying-circulating-micrornas-acute-myocardial

Research

JoVE Journal

Medicine

8.0K 조회수.  University of Freiburg. 순환 microRNAs는 심혈 관 질환 및 급성 심근 경색에 대 한 생체로 약속을 보여주었다. 이 연구에서 우리는 미르 추출, 반전 녹음 방송 및 심혈 관 질환 환자의 혈 청에서 miRNAs의 절대 정량화에 대 한 디지털 PCR 프로토콜을 설명합니다.

비디오: Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

고양이에서 급성 심근 경색

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

연구

의학

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

돼지의 심근 경색 및 기능적 결과의 평가

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

쥐 심근 경색을 유도 최소 침습 수술

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

마우스의 왼쪽 전방 내림차순 관상 동맥의 영구 결찰 : 심근 경색 리모델링과 심장 마비의 모델

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

급성 심근 경색의 돼지 모델의 주요 결과 평가

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

쥐의 급성 심근 경색의 실시간 압력-볼륨 분석

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

마우스에서 심근 경색을 연구하는 극저온 손상 모델

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

쥐의 심근 경색과 격리 된 작업 심장 시스템에 의한 심장 기능 평가를 유도하는 반 최소 침습 적 방법

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Murine 모델에서 심근 경색 후 MSC 로딩 주사용 하이드로겔의 자궁 내 이식

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...