우리가 실험에서 그녀의 도움에 대 한 미스 신 재미 우 감사 하고자 합니다. 이 연구는 장 공 의학 연구 프로그램 (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373)와 IRB 100-4470A3, 104-2462A3, 대만에서 교부 금에 의해 지원 되었다.

임상 실험실 개선 개정 (CLIA) 1988 년의 기관 검토 위원회의 장 궁 메모 리얼 병원에서 승인 하는 규정 요구 사항에 따라 실시 되었다 테스트 실험실에서 수행 하 고 대학 (라이센스 번호 100-4470A3 및 104-2462A3). 동의 모든 환자에서 얻은 했다.
1. 혈액 표본 수집
<ol><li>상업적으로 사용할 수 있는 치료를 EDTA 튜브로 전체 혈액을 수집 합니다. 부드럽게 혼합 하 고 처리까지 4 ° C에서 혈액 샘플을 저장 합니다.</li>
</ol>2. 말 초 혈액에서 DNA 추출
DNA 추출 키트를 사용 하 여 게놈 DNA 추출 ( 재료의 표참조).
<ol><li>4.0 mL 혈액 샘플을 솔루션 1의 10 mL를 추가 합니다. 10 분 동안 얼음에 샘플을 품 어.</li>
	<li>4 ° c.에서 5 분 동안 2000 x g에서 샘플을 원심 제거 하 고 상쾌한을 신중 하 게 삭제. 솔루션 1의 3 mL에 펠 릿을 resuspend.</li>
	<li>다시 4 ° C에서 5 분 동안 2000 x g에서 샘플을 원심 하 고 상쾌한 삭제. 솔루션 2의 3 mL에 펠 릿을 resuspend.</li>
	<li>100 µ g/mL의 최종 농도 고 잘 섞어 나 재고 솔루션 (225 mg/mL)의 10 µ L를 추가 합니다. 37 ° C에서 밤새 품 어.</li>
	<li>10 분 추가 0.8 mL 솔루션 3의 샘플에 대 한 얼음에 튜브를 진정 하 고 반전 3-5 번. 5 분 동안 얼음에 샘플을 놓습니다.</li>
	<li>4 ° c.에 15 분 동안 3000 x g에서 샘플을 원심 조심 스럽게 15 mL 원뿔 메 마른 분리기 관에는 상쾌한 전송.</li>
	<li>20 µ g/mL의 최종 농도를 RNase 재고 솔루션 (10 mg/mL)의 6 µ L를 추가 합니다. 15 분 동안 37 ° C에서 품 어.</li>
	<li>부드럽게 여러 번 DNA를 침전을 반전 하 여 이소프로필 알코올과 혼합의 2.5 mL를 철저 하 게 추가 하는 방법 (흰색, 부드러운 재질의 가닥 형성). 큰 구멍 피펫으로 사용 하 여 새로운 1.5 mL 원심 분리기 튜브 DNA 비례적 전송.</li>
	<li>4 ° c.에 3 분 동안 12000 x g에서 원심 분리기 상쾌한 신중 하 게 삭제 합니다. 씻어 DNA 펠 릿의 70% 에탄올 500 µ L을 추가 하 여.</li>
	<li>1 분 동안 12000 x g에서 centrifuge 고 상쾌한 삭제. 공기 건조 실 온에서 DNA 펠 릿. DdH2O의 100 µ L을 추가 하 고 65 ° C 15 분 DNA resuspend에서 품 어.</li>
</ol>3. 게놈 DNA 정량화
분 광 광도 계를 사용 하 여 ( 재료의 표참조) 게놈 DNA의 품질과 수량을 감지 하.
<ol><li>분 광 광도 계 컴퓨터에 연결 된 컴퓨터에 로그인 합니다. 소프트웨어를 엽니다.</li>
	<li>분 광 광도 계 기계 초기화:<ol>
<li>분 광 광도 계 소프트웨어에 "핵 산"을 클릭 합니다.</li>
		<li>일회용 종이 지우기와 순화 된 물 받침대를 청소.</li>
	</ol>
</li>
	<li>분 광 광도 계 빈:<ol>
<li>주 춧 대에 순화 된 물의 2 µ L을 로드 합니다.</li>
		<li>분 광 광도 계의 윗부분을 낮출 하 고 "빈" 보정을 클릭 합니다.</li>
		<li>완료 되 면 위 팔을 들어올린 닦기와 받침대를 건조.</li>
	</ol>
</li>
	<li>측정 샘플:<ol>
<li>샘플 종류에 "DNA 50"을 클릭 합니다.</li>
		<li>주 춧 대에 DNA 샘플의 2 µ L을 로드 합니다.</li>
		<li>낮은 팔 위쪽을 A260/A280 비율과 샘플의 농도 측정 하기 위해 "측정"을 클릭 합니다.</li>
		<li>지우기와 순화 된 물으로 각 실행 사이 받침대를 청소.</li>
	</ol>
</li>
	<li>"Print Screen" 데이터 수집을 클릭 합니다.</li>
</ol>4. 유전자 돌연변이 분석
PCR4대상 DNA를 증폭. 25 µ L 반응에서 PCR을 수행 DNA 중 합 효소를 사용 하 여 혼합 키트 ( 재료의 표참조). 표 1 에서 intronic 뇌관 TTR 유전자를 보여준다.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>유전자</td>
			<td>뇌관 순서</td>
		</tr>
<tr>
<td>Exon1</td>
			<td>F: 5'-TCAGATTGGCAGGGATAAGC-3' R: 5'-GCAAAGCTGGAAGGAGTCAC-3'</td>
		</tr>
<tr>
<td>Exon2</td>
			<td>F: 5'-TCTTGTTTCGCTCCAGATTTC-3' R: 5'-TCTACCAAGTGAGGGGCAAA-3'</td>
		</tr>
<tr>
<td>Exon3</td>
			<td>F: 5'-GTGTTAGTTGGTGGGGGTGT-3' R: 5'-TGAGTAAAACTGTGCATTTCCTG-3'</td>
		</tr>
<tr>
<td>Exon4</td>
			<td>F: 5'-GACTTCCGGTGGTCAGTCAT-3' R: 5'-GCGTTCTGCCCAGATACTTT-3'</td>
		</tr>
</tbody></table>표 1입니다. TTR 유전자 intronic 뇌관. (NCBI 참고 순서: NC_000018.10)
<ol><li>0.2 mL PCR 튜브를 표 2 에 시 약을 추가 합니다.</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>구성 요소</td>
			<td>볼륨 (µ L)</td>
			<td>최종 농도</td>
		</tr>
<tr>
<td>10 x 버퍼</td>
			<td>2.5</td>
			<td>1 x</td>
		</tr>
<tr>
<td>MgCl2 (25 mM)</td>
			<td>1.5</td>
			<td>1.5 m m</td>
		</tr>
<tr>
<td>360 GC 증강</td>
			<td>1</td>
			<td>-</td>
		</tr>
<tr>
<td>360 DNA 중 합 효소</td>
			<td>0.2</td>
			<td>2 U/반응</td>
		</tr>
<tr>
<td>dNTP 믹스 (각 25 mM)</td>
			<td>2</td>
			<td>2 m m</td>
		</tr>
<tr>
<td>뇌관 F (10 µ M)</td>
			<td>1</td>
			<td>0.4 Μ M</td>
		</tr>
<tr>
<td>뇌관 R (10 µ M)</td>
			<td>1</td>
			<td>0.4 Μ M</td>
		</tr>
<tr>
<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
<tr>
<td>PCR-등급 물</td>
			<td>13.8</td>
			<td>-</td>
		</tr>
<tr>
<td>총 볼륨</td>
			<td>25</td>
			<td>-</td>
		</tr>
</tbody></table>표 2입니다. PCR 반응 조건입니다.
<ol start="2"><li>부드럽게 혼합 하 고 짧게 튜브의 하단에 모든 구성 요소를 수집 하는 벤치탑 원심 분리기에 원심. 튜브는 thermocycler 장소.</li>
	<li>30 사이클에 대 한 PCR을 수행 합니다. 각 주기 30 변성 단계 이루어져 94 ° C, 30에 대 한 단련 하는 뇌관에서 s 58 ° C, 및 45에 대 한 중 합 효소 확장에서 s s 72 ° C에서 ( 표 3참조).</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>단계</td>
			<td>온도 (° C)</td>
			<td>시간</td>
			<td>사이클</td>
		</tr>
<tr>
<td>초기 변성</td>
			<td>95</td>
			<td>5 분</td>
			<td>1</td>
		</tr>
<tr>
<td>DNA 변성 단계</td>
			<td>94</td>
			<td>30 s</td>
			<td>30 사이클</td>
		</tr>
<tr>
<td>뇌관 어 닐 링의 단계</td>
			<td>58</td>
			<td>30 s</td>
			<td>30 사이클</td>
		</tr>
<tr>
<td>중 합 효소 확장 단계</td>
			<td>72</td>
			<td>45 s</td>
			<td>30 사이클</td>
		</tr>
<tr>
<td>포스트 신장 단계</td>
			<td>72</td>
			<td>10 분</td>
			<td>1</td>
		</tr>
<tr>
<td>PCR의 끝 자전거</td>
			<td>4</td>
			<td>무기한</td>
			<td></td>
		</tr>
</tbody></table>표 3입니다. 사이클링 증폭 PCR 제품을 위한 조건.
<ol start="4"><li>젤 전기 이동 법으로 시각화 하 여 PCR 제품을 확인:<ol>
<li>1.2 %agarose 젤 준비:<ol>
<li>0.5의 100 mL의 agarose 분말 1.2 g 혼합 microwavable 플라스 크에 x TBE. 전자 레인지는 agarose는 완전히 용 해 될 때까지 2 분. 55 ° c agarose 혼합물을 진정</li>
			<li>Ethidium 평범한 사람 해결책 (10mg/mL)의 2 µ L agarose 혼합물에 추가 하 고 부드럽게 혼합. 약 5-7 m m 젤 트레이에 agarose 혼합물을 부 어 다음 장소에는 comb(s)를 추가 합니다. (적어도 30 분)을 공고히 하 고 comb(s)를 조심 스럽게 제거를 하실 수 있습니다.</li>
		</ol>
</li>
		<li>샘플을 로드 하 고 실행 하는 agarose 젤:<ol>
<li>트레이 젤 전기 이동 법 셀에 놓습니다. 0.5 전기 셀 채우기 x TBE 젤 커버까지.</li>
			<li>신중 하 게 로드 2 µ L 100 bp DNA의 분자 크기 표식으로 젤의 한 차선에 ( 재료의 표참조) 사다리. 부하 염료/샘플 혼합 (5 µ L PCR 제품 믹스의 로드 염료 x 1 µ L 6와) 추가 차선에.</li>
			<li>전원 공급 장치를 켭니다. 100 V에서 25 분 젤을 실행 합니다.</li>
			<li>젤 전기 이동 법 셀에서 제거 합니다. 자외선 이미징 시스템에서에서 DNA 파편을 시각화.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. DNA 시퀀싱
<ol><li>상업 키트를 사용 하 여 PCR 제품을 정화 하 고 상업적으로 또는 자동 시퀀서는 시퀀싱 ( 재료의 표참조).</li>
	<li>뉴클레오티드 시퀀스 뉴클레오티드 데이터베이스10뉴클레오티드 쿼리를 비교 하는 NCBI 폭발 서버에 제출 합니다. 결과 표시 후 시퀀스 정렬을 수행 하 고 예상된 돌연변이 식별 합니다.</li>
</ol>

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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=Clinical+diagnosis+and+typing+of+systemic+amyloidosis+in+subcutaneous+fat+aspirates+by+mass+spectrometry-based+proteomics.">Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics.</a> Haematologica. 99 (7), 1239-1247 (2014).</li><li>Nakamura, M., 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+new+transthyretin+variant+(Ile49)+in+familial+amyloidotic+polyneuropathy+using+electrospray+ionization+mass+spectrometry+and+nonisotopic+RNase+cleavage+assay.">Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay.</a> Human Heredity. 49 (4), 186-189 (1999).</li><li>Ueda, M., Ando, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+transthyretin+amyloidosis+therapy.">Recent advances in transthyretin amyloidosis therapy.</a> Translational Neurodegeneration. 3, 19 (2014).</li><li>Brown, E. E., 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=Genetic+testing+improves+identification+of+transthyretin+amyloid+(ATTR)+subtype+in+cardiac+amyloidosis.">Genetic testing improves identification of transthyretin amyloid (ATTR) subtype in cardiac amyloidosis.</a> Amyloid. 24 (2), 92-95 (2017).</li><li>Tasaki, M., 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=Rapid+detection+of+wild-type+and+mutated+transthyretins.">Rapid detection of wild-type and mutated transthyretins.</a> Annals of Clinical Biochemistry. 53 (4), 508-510 (2015).</li><li>Plante-Bordeneuve, V., 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=Diagnostic+pitfalls+in+sporadic+transthyretin+familial+amyloid+polyneuropathy+(TTR-FAP).">Diagnostic pitfalls in sporadic transthyretin familial amyloid polyneuropathy (TTR-FAP).</a> Neurology. 69 (7), 693-698 (2007).</li><li>Hsu, J. 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=A+prospective,+observational+study+of+patients+with+uncommon+distal+symmetric+painful+small-fiber+neuropathy.">A prospective, observational study of patients with uncommon distal symmetric painful small-fiber neuropathy.</a> PLoS ONE. 12 (9), 0183948 (2017).</li><li>Salvi, F., Pastorelli, F., Plasmati, R., Bartolomei, I., Dall'Osso, D., Rapezzi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+and+phenotypic+correlation+in+an+Italian+population+of+hereditary+amyloidosis+TTR-related+(HA-TTR):+Clinical+and+neurophysiological+aids+to+diagnosis+and+some+reflections+on+misdiagnosis.">Genotypic and phenotypic correlation in an Italian population of hereditary amyloidosis TTR-related (HA-TTR): Clinical and neurophysiological aids to diagnosis and some reflections on misdiagnosis.</a> Amyloid. 19, 58-60 (2012).</li><li>Suhr, O. B., Lundgren, E., Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mutation,+two+distinct+disease+variants:+Unravelling+the+impact+of+transthyretin+amyloid+fibril+composition.">One mutation, two distinct disease variants: Unravelling the impact of transthyretin amyloid fibril composition.</a> Journal of Internal Medicine. 281 (4), 337-347 (2017).</li><li>Vil&#224;-Rico, M., 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=Quantitative+analysis+of+post-translational+modifications+in+human+serum+transthyretin+associated+with+familial+amyloidotic+polyneuropathy+by+targeted+LC-MS+and+intact+protein+MS.">Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS.</a> Journal of Proteomics. 127, 234-246 (2015).</li></ol>

저자는 공개 없다.

프로토콜 내에서 두 가지 중요 한 단계가 있습니다. 첫째, 백혈구 세포의 충분 한 수를 위해, hemodiluted 견본 피할된11이어야 한다. 둘째, 적절 한 PCR 뇌관의 사용이 이다 신뢰할 수 있는 결과12를 기본. 디자인 뇌관4,13; 뇌관 폭발 웹 도구를 사용 하는 우리 4 개의 TTR exons의 각 측에 40의 기본적인 쌍의 최소 처리 되어야 합...

Transthyretin (TTR) amyloidosis (ATTR) 조직의 유전 amyloidosis1, 가장 일반적인 형태 이며 transthyretin (TTR) 유전자2상 염색체 지배적 상속 된 돌연변이 의해 발생할 수 있습니다. TTR 돌연변이 tetrameric 단백질 구조를 불안정 하 게 하 고 녹말 체 소2로 재어셈블합니다 단위체로 그것의 분리 이어질. 100 amyloidogenic TTR 변이 보고 전세계1되었습니다. 중 합 효소 연쇄 반응 (PCR) 방법으로 유전자 분석 TTR 점 돌연변이의 존재를 확인 하 고 피하는 남쪽 오 점3에 비해 방사성 레이블이 프로브의 처리를 포함 하는 장점이 있다. PCR은 현대 과학4에 수많은 분야에 적용 된, 쉽게, 저렴 한, 빠르고 신뢰할 수 있는 기술입니다.
이 진보 및 치명적인 질병의 조기 진단의 phenotypic이 주어진 도전 이다. 관심과 ATTR 신흥 치료5의 초기 발현에 광범위 한 인식 증가, TTR 유전자 검사를 포함 한 적절 한 진단 연구는 따라서 비판적으로 예 후를 개선 하기 위해 근본적인. 또한, 다른 변이 특성의 다른 penetrance, 발병 연령, 진행, 질병의 심각도, 메디아 생존, 간 이식, 또는 TTR 안정기2,6, 효능의 패턴에 연관 된 고 가변도 신경학 적인 참여의 유전 상담7,,89에 대 한 큰 의미를가지고. 게다가, 매우 정확한 유전자 검사는 두 가지 유형의 속성을 구분 하는 유일한 도구: 유전 (돌연변이) 및 야생 유형 (비 돌연변이 형태, 치 조직의 amyloidosis, 특수)7. 그것은 치료2넓게 변화 하기 때문에, 속성의 종류를 확인 하는 것이 필수적입니다. 그러므로, TTR 유전자 검사의 stepwise 프로토콜을 설명 하는 증가 필요는 없다.
돌연변이 검출 하는 분자 접근 방식은 예를 들어 Ala97Ser, 대만, 가장 일반적인 발병 변이 사용 하 여 그림 것입니다. 수정 DNA 추출 단계에서 사용 되는 세 가지 솔루션의 양을 줄일 하 고 충분 한 양의 DNA 생성 합니다. 이 프로토콜에서 모든 4 개의 TTR exons 지역 5' 상류 3' 하류, 발기인, introns를 포함 하 여 동안, 분석 되었다 그리고 번역 되지 않은 영역 (UTR) 시퀀싱 하지 했다.

<table border="0" cellpadding="0" cellspacing="0" style="width:951px;" width="951">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">EDTA-treated tubes</td>
			<td style="width:199px;">BD</td>
			<td style="width:347px;"></td>
			<td style="width:192px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA Extraction Kit</td>
			<td style="width:199px;">Stratagen</td>
			<td style="width:347px;">200600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">NanoDrop ND2000 spectrophotometer&#160;</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:347px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Delicate Task Wipers</td>
			<td style="width:199px;">Kimberly-Clark</td>
			<td style="width:347px;">Kimtech Science Kimwipes</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:213px;">AmpliTaq Gold 360 DNA Polymerase kit</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">4398823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">TTR gene intronic primers&#160;</td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">F: 5&#8217;-TCAGATTGGCAGGGATAAGC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon1</td>
			<td style="width:347px;">R: 5&#8217;-GCAAAGCTGGAAGGAGTCAC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">F: 5&#8217;-TCTTGTTTCGCTCCAGATTTC-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon2</td>
			<td style="width:347px;">R: 5&#8217;-TCTACCAAGTGAGGGGCAAA-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">F: 5&#8217;-GTGTTAGTTGGTGGGGGTGT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon3</td>
			<td style="width:347px;">R: 5&#8217;-TGAGTAAAACTGTGCATTTCCTG-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">F: 5&#8217;-GACTTCCGGTGGTCAGTCAT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;"></td>
			<td style="width:199px;">Exon4</td>
			<td style="width:347px;">R: 5&#8217;-GCGTTCTGCCCAGATACTTT-3&#8217;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">thermocycler</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">GeneAmp PCR System 9700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">electrophoresis cell&#160;</td>
			<td style="width:199px;">ADVANCE</td>
			<td style="width:347px;">Mupid-2plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">DNA ladder</td>
			<td style="width:199px;">Protech</td>
			<td style="width:347px;">PT-M1-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">dye</td>
			<td style="width:199px;">BioLabs</td>
			<td style="width:347px;">B7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">AlphaImager EC</td>
			<td style="width:199px;">Alpha Innotech</td>
			<td style="width:347px;">AlphaImager EC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">automatic sequencer&#160;</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:347px;">3730xl DNA Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

유전자 검사 이며 유전 transthyretin에 대 한 가장 신뢰할 수 있는 테스트 관련 amyloidosis transthyretin amyloidosis (ATTR)의 대부분의 경우에서 수행 되어야 합니다. 속성은 유형이 다른 고기;와 희귀 하지만 치명적인 질병 따라서, 진단 때로는 지연 됩니다. 관심과 ATTR 신흥 치료의 초기 발현에 광범위 한 인식 증가 함께 transthyretin (TTR) 유전자를 포함 하 여 적절 한 진단 연구, 테스트, 종류를 확인 하 고 ATTR의 변종 따라서 예 후를 개선 하기 위해 근본적인. 중 합 효소 연쇄 반응 (PCR) 방법으로 유전자 분석 TTR 점 돌연변이의 존재를 훨씬 더 신속 하 게 확인 하 고 남쪽 오 점 등 기존 방법 보다 안전. 여기, 우리가 ATTR Ala97Ser 돌연변이, 대만에서 가장 일반적인 발병 돌연변이의 유전자 확인을 보여 줍니다. 프로토콜 구성 4 가지 주요 단계: 수집 전 혈 견본, DNA 추출, PCR와 모든 4 개의 TTR exons의 유전자 분석 및 DNA 연속.

Agarose 젤 전기 이동 법 두 환자와 exon TTR 유전자 (그림 1)의 4에 대 한 454 bp의 PCR 제품을 포함 한 예상된 크기에의 한 건강 한 개별 계시 밴드.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> 그림 1 . 젤 전기 이동 법 묘사 PCR 증폭 TTR 유전자. 일반: 건강 한 ?...

Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

여기, 우리는 예를 들어 Ala97Ser, 대만, 가장 일반적인 발병 변이 사용 하 여 유전 transthyretin amyloidosis의 진단에 대 한 점 돌연변이의 존재를 확인 하는 프로토콜 제시.

유전 Transthyretin Ala97Ser의 유전 분석 관련 Amyloidosis

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

genetic-analysis-hereditary-transthyretin-ala97ser-related

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Medicine

7.6K 조회수.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. 여기, 우리는 예를 들어 Ala97Ser, 대만, 가장 일반적인 발병 변이 사용 하 여 유전 transthyretin amyloidosis의 진단에 대 한 점 돌연변이의 존재를 확인 하는 프로토콜 제시.

비디오: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

FISH for Pre-implantation Genetic Diagnosis

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

사전 이식 유전자 진단 생선

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

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Examining the Characteristics of Episodic Memory using Event-related Potentials in Patients with Alzheimer's Disease

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic mild cognitive impairment (aMCI) and Alzheimer's disease (AD). Whereas behavioral data inform us about the patients' performance, ERPs allow us to record discrete changes in brain activity. Further, ERPs can give us insight into the onset, duration, and interaction of independent cognitive processes associated with memory retrieval. In patient populations, these types of studies are used to examine which aspects of memory are impaired and which remain relatively intact compared to a control population. The methodology for collecting ERP data from a vulnerable patient population while these participants perform a recognition memory task is reviewed. This protocol includes participant preparation, quality assurance, data acquisition, and data analysis. In addition to basic setup and acquisition, we will also demonstrate localization techniques to obtain greater spatial resolution and source localization using high-density (128 channel) electrode arrays.

The methodology for collecting high-density event-related potential data while patients with Alzheimer's disease perform a recognition memory task is reviewed. This protocol will include subject preparation, quality assurance, data acquisition, and data analysis.

알츠하이머 병 환자에서 이벤트 관련 후보를 사용하여 에피소드 메모리의 특성을 검토

Our laboratory uses event-related EEG potentials (ERPs) to understand and support behavioral investigations of episodic memory in patients with amnestic ...

Identification of Disease-related Spatial Covariance Patterns using Neuroimaging Data

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components (Figure 1). Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions. Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors7,8. Using logistic regression analysis of subject scores (i.e. pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. composite networks with improved discrimination of patients from healthy control subjects5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation11. These standardized values can in turn be used to assist in differential diagnosis12,13 and to assess disease progression and treatment effects at the network level7,14-16. We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease.

Multivariate techniques including principal component analysis (PCA) have been used to identify signature patterns of regional change in functional brain images. We have developed an algorithm to identify reproducible network biomarkers for the diagnosis of neurodegenerative disorders, assessment of disease progression, and objective evaluation of treatment effects in patient populations.

신경 영상 데이터를 사용하여 질병과 관련된 공간 공분산 패턴의 식별

The scaled subprofile model (SSM)1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

마우스에서 관절염 통증의 Monoiodoacetate 모델

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

마우스에서 가속 죽상 경화증의 유도: "와이어 부상" 모델

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

검색 및 대 식 세포 관련 Murine 창 자 염증의 정량화에 대 한 단층 형광 중재

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

Intravital 현미경 Monocyte 유도 및 주변 동맥 질병의 Murine 모델에서 종양 관련 신생의

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Comparing Bibliometric Analysis Using PubMed, Scopus, and Web of Science Databases

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly on life sciences and biomedical disciplines, whereas Scopus and Web of Science are multidisciplinary. The protocol described in the current study was used to search for publications from Jordanian authors in the years 2013-2017. In this protocol, how to use each database to conduct this type of search is explained in detail. A Scopus search resulted in the highest number of documents (11,444 documents), followed by a Web of Science search (10,943 documents). PubMed resulted in a smaller number of documents due to its narrower scope and coverage (4,363 documents). The results also show a yearly trend in: (1) the number of publications, (2) the disciplines that have the most publications, (3) the countries of collaboration, and (4) the number of open access publications. In contrast, PubMed has a sophisticated keyword optimization service (i.e., Medical Subject Heading, or MeSH), while both Scopus and Web of Science provide search analysis tools that can produce representative figures. Finally, the features of each database are explained in detail and several indices that can be extracted using the search results are provided. This study provides a base for using literature databases for bibliometric analysis.

Literature databases are commonly used to assess publications in a certain subject, discipline, country, or region of the world, a practice known as bibliometric analysis. The current protocol details how to use PubMed, Scopus, and Web of Science databases to do bibliometric analysis.

PubMed, 스코퍼스 및 과학 데이터베이스 웹을 사용한 참고 문헌 분석 비교

Literature databases (i.e., PubMed, Scopus, and Web of Science) differ in terms of their coverage, focus, and the tool they provide. PubMed focuses mainly ...

Integrating Augmented Reality Tools in Breast Cancer Related Lymphedema Prognostication and Diagnosis

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients subjected to axillary surgery and/or radiations. Its etiology is multifactorial and include also tumor-specific pathological features, such as lymphovascular invasion (LVI) and extranodal extension (ENE). To date, no widely employed guidelines for the early diagnosis of BCRL are available. Here, we illustrate a protocol for a digitally assisted BCRL assessment using a 3D laser scanner (3DLS) and a tablet computer. It has been specifically optimized in a discovery cohort of high-risk breast cancer patients. This study provides a proof-of-principle that augmented reality tools, such as 3DLS, can be incorporated into the clinical workup of BCRL to allow for a precise, reproducible, reliable, and cheap diagnosis.

Breast cancer related lymphedema is frequent in breast cancer survivors, but there are not widely employed guidelines for its diagnosis and quantification. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare upper limb volume in breast cancer patients.

유방암 관련 림프부종 예후 및 진단에 증강 현실 도구를 통합

Breast cancer related lymphedema (BCRL) is a detrimental condition characterized by fluid accumulation in the upper limb in breast cancer patients ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

래트 무릎의 순환 로딩 유도 관절내 연골 병변 모델을 생성하기 위한 비침습적 방법

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...