Name: serum and plasma copy number detection using real-time pcr
Uploaded: 2017-12-15T14:00:00.000+00:00
Duration: 9 min 21 s
Description: Watch this Scientific Journal Video about Serum and Plasma Copy Number Detection Using Real-time PCR at JoVE.com

Biz Chiara Molinari ve Filippo Martignano veri analizi desteği için teşekkür ederim.

DNA izolasyonu kopya numara analiz için gerçek zamanlı PCR gerçekleştirmek için serum veya plazma numuneleri, protokol oluşur. DNA ekstraksiyon, DNA miktar denetimi (Spektrofotometre) ve gerçek zamanlı PCR belirli hedefleri için yapıldı. Şekil 1' de, zaman çizgisi ve yordamlar bir özetini bildirilir.
Protokol IRST insan araştırma Etik Komitesi kuralları izler.
1. serum toplama ve işleme
<ol>
	<li>Yaklaşık 5 mL tam kan serum elde etmek için antikoagülan olmadan bir serum tüp toplamak. Kan tüpleri 4 ° C'de işleme kadar korumak.</li>
	<li>Santrifüj tüpleri, 1000 x g 4 ° C'de 15 dakika</li>
	<li>Dikkatle serum 2 mL tüpler içine aktarın.</li>
	<li>Toplama tüp atmak ve hemen süpernatant-80 ° c kadar kullanmak dondurma.</li>
</ol>
2. plazma toplama ve işleme
<ol>
	<li>Yaklaşık 10 mL tam kan plazma elde etmek için EDTA ile plazma tüp içinde toplamak. Tüp tamamen doldurmak ve sonra 8 kere tersine çevirin. Kan tüpleri 4 ° C'de işleme kadar korumak.</li>
	<li>Tüp 1800 x g oda sıcaklığında 15 dakika santrifüj hiçbir fren ayarı ile santrifüj kapasitesi.</li>
	<li>Dikkatle plazma üst kısmındaki 2 mL tüpler içine aktarın. En az 1 mL süpernatant beyaz hücre katmanı üzerinde bırakarak transferi, tekrarlayın. Not: üst kısmındaki süpernatant aktarma hücreleri veya hücre artıkları bulaşma riskini azaltır.</li>
	<li>Toplama tüp atmak ve hemen süpernatant-80 ° c kadar kullanmak dondurma.</li>
</ol>
3. Serum veya plazma DNA izolasyonu
Not: Aşağıdaki adımlarda modifiye ticari iletişim kuralını kullanarak serum veya plazma DNA izolasyonu uygulanmalıdır.
<ol>
	<li>(0,5 2 mL içeren) bir aliquot serum veya plazma oda sıcaklığında erimek.</li>
	<li>Girdap ve örnek mix ve 0.5 mL serum veya plazma net 1.5 mL tüp içine aktarın. Geri kalan malzeme-80 ° C'de dondurmak</li>
	<li>50 µL İndinavir k (20 mg/mL) doğrudan örneğine ekleyin.</li>
	<li>0.5 mL lizis arabelleği için örnek ve de pipetting tarafından karıştırın.</li>
	<li>Tüpler kapatın ve örnekleri 56 ° c ısı blok 15 dk için kuluçkaya.</li>
	<li>% 100 etanol belirtilmiş miktarı yıkama arabellek 1 ve 2, üreticinin yönergelerini tarafından belirtildiği şekilde eklenir.</li>
	<li>Örnekleri geri oda sıcaklığına getirmek ve mutlak etanol 0.5 mL ve de pipetting tarafından karıştırın.</li>
	<li>650 µL adım 3.7 ayrılık sütun ve 1 dk. için 6.000 x g, santrifüj elde karışımı ekleyin.</li>
	<li>Aracılığıyla akış içeren tüp atın ve sütun yeni bir temiz toplama tüp yerleştirin. Adım 3.8 ve 3,9 bir kez daha yineleyin.</li>
	<li>500 µL yıkama tampon 1, sütun ve 1 dk. için 6.000 x g, santrifüj kenarında ıslatmadan ekleyin.</li>
	<li>Aracılığıyla akış içeren tüp atın ve eski yerine koymak o ile yeni bir temiz toplama tüp.</li>
	<li>500 µL yıkama tampon 2, sütun ve 3 dak için tam hızda (20.000 x g) santrifüj kenarında ıslatmadan ekleyin.</li>
	<li>Aracılığıyla akış içeren tüp atın ve eski yerine koymak o ile yeni bir temiz toplama tüp.</li>
	<li>Herhangi bir kalıntı çamaşır arabellek kaldırmak için 3 dakika için tam hızda (20.000 x g) santrifüj kapasitesi.</li>
	<li>Sütun bir temiz 1.5 mL tüp içine yerleştirin. 150 µL elüsyon arabelleği ekleyin ve bu arabellek sütun ıslatıyor emin olmak için 7 dk. bekleyin.</li>
	<li>1 dk. için 8000 x g de santrifüj kapasitesi.</li>
	<li>Elute DNA'ın en büyük kurtarma sağlamak 1 dk için tekrar aynı sütun ve santrifüj tam hızda (20.000 x g) içine 3,16 adım pipette.</li>
</ol>
4. DNA miktar ve seyreltme
<ol>
	<li>Miktar DNA bir Spektrofotometre kullanarak gerçekleştirin. Örnek 2 µL tezgah üstü Spektrofotometre başına üreticinin yönergelerini kullanın. Not: Eğer DNA miktar gerçek zamanlı PCR ile devam etmek yeterli değildir (en az 120 ng), yeni bir DNA izolasyon işlemi gerçekleştirin.</li>
	<li>DNA için son hacim (yaklaşık 50 µL) tüm gerçek zamanlı PCR yeterli 2.5 ng/µL serum veya plazma sulandırmak.</li>
</ol>
5. gerçek zamanlı PCR
<ol>
	<li>Tezcan kopya numarası deneyleri, referans gen tahlil, seyreltilmiş DNA örnekleri ve buzun içinde havuzlu KALİBRATÖRLER. Oda sıcaklığında 4 ° C'de depolanan ana mix equilibrate</li>
	<li>Hedef tahlil, referans gen testin 1 µL ve ana Mix 10 µL 1 µL karışımı her örnek ve KALİBRATÖRLER havuzlu 3 çoğaltır için hazırlar. Negatif kontrol (su) ve 2 ilave örnekler de dahil olmak üzere karışımı hazırlayın.</li>
	<li>96'plaka, aliquot 12 µL her kuyuya karışımı iyi. Pipet ya da spin tüpleri var.</li>
	<li>Her DNA örneği ve her kuyuya havuzlu KALİBRATÖRLER 8 µL (2.5 ng/µL konsantrasyon) ekleyin. Pipet ya da spin tüpleri var. Her şey için belgili tanımlık uç değiştirin. Not: 96-şey plaka kullanarak gerçek zamanlı her deneme için 30 DNA örnekleri işlenebilir: 30 x 3 örnekleri + 1 x 3 KALİBRATÖRLER havuza alınan + negatif kontrol 94 =.</li>
	<li>Kısaca spin plaka 1000 x g de aşağı.</li>
	<li>Aşağıdaki iletişim kuralını kullanarak plakayı araştırmanı: 40 devredir 15 95 ° C'de 10 dakika, 95 ° C'de sahne tutun s ve 60 ° C için 1 dakika.</li>
</ol>
6. veri analizi ve yorumu
<ol>
	<li>Amplifikasyon arsa sonuçları altındaki "seçeneği" bölümünde, analiz ilk hedef gen için 0.2 Ct eşik ayarlayın. Kümedeki her hedef veya başvuru genler için yineleyin. .Txt uzantısı gerçek zamanlı PCR dosyasında dışa aktarmak için araç çubuğu'na "Dışa Aktar" tuşuna basın. Sadece "sonuçlar" Seç bayrak → verilecek veriyi uzantısı .txt → basın "start verme" → yakın dosya türü olarak verme aracını seçin.</li>
	<li>Analiz yazılımı açın ve dosyayı .txt (araç çubuğu → alma) alın.</li>
	<li>Analiz ve araç çubuğu (oyun sembol) tarafından analiz ayarı seçmek için dosya adını seçin.</li>
	<li>Kalibratör örnek ve hedef (Örneğin, 1 AR gen için kopya) kopya bilinen sayısını belirtin. Basın "uygulanır."</li>
	<li>Her örnek için farklı delta Ct (ΔCt) ile karşılaştırıldığında diğer wells ile bir şey atlayın.</li>
	<li>Deney (basın oyun sembol) yeniden çözümle.</li>
	<li>Arsa veya tablo bar kopya sayısıyla sonuçlarını değerlendirmek. Not: Güven, Z-score ve sonuçları yorumlama için yararlı olabilir analiz çoğaltır gibi çok sayıda parametre sonuçları tablo içerir.</li>
</ol>

<ol>
	<li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+as+a+diagnostic+marker+for+cancer:+current+insights.">Cell-free DNA as a diagnostic marker for cancer: current insights.</a> Onco Targets Ther. 9, 6549-6559 (2016).</li><li>Heitzer, E., Ulz, P., Geigl, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+as+a+liquid+biopsy+for+cancer.">Circulating tumor DNA as a liquid biopsy for cancer.</a> Clin. Chem. 61 (1), 112-123 (2015).</li><li>Murtaza, 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=Non-invasive+analysis+of+acquired+resistance+to+cancer+therapy+by+sequencing+of+plasma+DNA.">Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA.</a> Nature. 497 (7447), 108-112 (2013).</li><li>Heitzer, E., Ulz, P., Geigl, J. B., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+genome-wide+somatic+copy+number+alterations+by+liquid+biopsies.">Non-invasive detection of genome-wide somatic copy number alterations by liquid biopsies.</a> Mol. Oncol. 10 (3), 494-502 (2016).</li><li>Diaz, L. 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=The+molecular+evolution+of+acquired+resistance+to+targeted+EGFR+blockade+in+colorectal+cancers.">The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.</a> Nature. 486 (7404), 537-540 (2012).</li><li>Dawson, S. J., 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=Analysis+of+circulating+tumor+DNA+to+monitor+metastatic+breast+cancer.">Analysis of circulating tumor DNA to monitor metastatic breast cancer.</a> N. Engl. J. Med. 368 (13), 1199-1209 (2013).</li><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+cell-free+AR+and+CYP17A1+copy+number+variations+may+associate+with+outcome+of+metastatic+castration-resistant+prostate+cancer+patients+treated+with+abiraterone.">Circulating cell-free AR and CYP17A1 copy number variations may associate with outcome of metastatic castration-resistant prostate cancer patients treated with abiraterone.</a> Br. J. Cancer. 112 (10), 1717-1724 (2015).</li><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+AR+copy+number+and+outcome+to+enzalutamide+in+docetaxel-treated+metastatic+castration-resistant+prostate+cancer.">Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer.</a> Oncotarget. 7 (25), 37839-37845 (2016).</li><li>Romanel, 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=Plasma+AR+and+abiraterone-resistant+prostate+cancer.">Plasma AR and abiraterone-resistant prostate cancer.</a> Sci. Transl. Med. 7 (312), (2015).</li><li>Conteduca, 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=Androgen+receptor+gene+status+in+plasma+DNA+associates+with+worse+outcome+on+enzalutamide+or+abiraterone+for+castration-resistant+prostate+cancer:+a+multi-institution+correlative+biomarker+study.">Androgen receptor gene status in plasma DNA associates with worse outcome on enzalutamide or abiraterone for castration-resistant prostate cancer: a multi-institution correlative biomarker study.</a> Ann Oncol. , (2017).</li><li>Attard, G., Antonarakis, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+cancer:+AR+aberrations+and+resistance+to+abiraterone+or+enzalutamide.">Prostate cancer: AR aberrations and resistance to abiraterone or enzalutamide.</a> Nat. Rev. Urol. 13 (12), 697-698 (2016).</li><li>Lee, T. H., Montalvo, L., Chrebtow, V., Busch, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+genomic+DNA+in+plasma+and+serum+samples:+higher+concentrations+of+genomic+DNA+found+in+serum+than+in+plasma.">Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma.</a> Transfusion. 41 (2), 276-282 (2001).</li><li>Chan, K. C., Yeung, S. W., Lui, W. B., Rainer, T. H., Lo, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+preanalytical+factors+on+the+molecular+size+of+cell-free+DNA+in+blood.">Effects of preanalytical factors on the molecular size of cell-free DNA in blood.</a> Clin. Chem. 51 (4), 781-784 (2005).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarının bildirin.

AR Serum ve plazma örnek analizde CN stratifikasyon kastrasyon dayanıklı prostat kanseri (CRPC) hastaların için yeni, non-invaziv bir yaklaşım temsil eder. Son zamanlarda AR CN abiraterone ve enzalutamide, tedavi öncesi ve sonrası kemoterapi7,8,9,10CRPC hastalarda sonuçlar tahmin etmek mümkün olduğunu kanıtlanmıştır.
Bizim yaklaşı...

Hücre ücretsiz DNA (cfDNA) kan dolaşan biyolojik kanser tanısı, prognoz, izleme ve tedavi direnci1,2tahmini için en iyi bir kaynağı olmak kanıtlanmıştır. Birçok çalışma DNA değişiklikler (mutasyonlar, kopya numarası varyasyonları, dokularda epigenetik değişiklikler) ve tümör DNA (ctDNA) dolaşan olduğunu teyit karşılık gelen plazma örnekleri1' de bulunanlar arasında iyi bir uyum göstermiştir birincil ve metastatik tümör doku değişiklikleri3için bilgilendirici. CtDNA eğitim imkanı böylece genomik düzenlemeler ve kopya numarası varyasyonları (CNVs) belirli oncogenes4potansiyel metastatik klonal ve subclonal hücreleri tanımlayan, yeniden inşası için izin verir. CtDNA belirli mutasyonlar ve özel hedeflenmiş tedaviler5,-6ile ilgili CNVs, limanlar gibi özellikle kanser tedavi izlemek için klinik olarak yararlı olabilir göstermiştir. Ayrıca doku biyopsisi ihtiyacını üstesinden gelir ve non-invaziv bir şekilde belirli kanser tedavisi sırasında farklı zamanlarda elde edilecek sonuçlar sağlar.
Prostat kanseri, boş hücre androjen dolaşan arasında önemli bir bağıntı ile ilgili olarak reseptör (AR) CNVs ve tedavi yanıt-e doğru abiraterone ve enzalutamide gösterilen, gösteren AR Gen kopya numarası (CN) cfDNA-ebilmek var olmak bir umut verici biyomarker tedavi direnci7,8,9,10,11tahmin yeteneğine sahip. CNVs ctDNA belirli genlerin değerlendirilen farklı duyarlılık, maliyet ve hız ile farklı yaklaşımlar kullanarak (örn. gerçek zamanlı, dijital PCR ve sonraki nesil sıralama).
Burada serum ve plazma örnekleri7,8cfDNA AR CN değerlendirmek için gerçek zamanlı PCR teknolojisinde çift yönlü deneyleri temel basit ve hızlı bir yaklaşım açıklar. Biz iki farklı genomik Berisi Bölgeler Klasiği intron 5 AR (Xq12) ve iki diğer genler, prostat kanseri (RNaseP, normal kopya numarası durumundaki bilinen iç standart referans genler olarak tasarlanmış iki farklı PCR deneyleri kabul 14q11 üzerinde bulunan; 1'de yer alan önce1, p 34). Biz bir duyarlık ve sonuçları duyarlılığını artırmak için yerine, iki referans genler seçildi. Bir DNA miktar olan 60 ng her tahlil birleşimi (kombine tahlil ve AR-assay_2 + AGO1 AR-assay_1 + RNaseP için) için güçlendirilmiş. Sağlıklı erkek üç serum veya plazma DNA örnekleri havuza alınmış ve bir Kalibratör kullanılır. Biz cutoffs, kabul &#62; AR kazanç için 1.5 ve &#60; 0,5 silinmek. Bu yöntemin ana avantajlarından biri bu esnek ve diğer genler Ayrıca, standart iç başvuru genler, tümör tipi ve özellikleri temelinde değiştirme değerlendirilebilir olduğunu olmasıdır.

<table><tbody><tr><td>BD Vacutainer Serum Tubes</td><td>Becton Dickinson</td><td>367814</td><td>whole blood tube for serum</td></tr><tr><td>BD Vacutainer EDTA Tubes</td><td>Becton Dickinson</td><td>366643</td><td>whole blood tube for plasma</td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td></td><td>the user could use also other companies</td></tr><tr><td>TaqMan Copy Number assay</td><td>Thermo Fisher Scientific</td><td>4400291</td><td>Pre-designed and validated assays with FAM-dye. We used the followig assay: AR_assay1: hs04107225 adn AR_assay2: hs04511283</td></tr><tr><td>TaqMan Copy Number assay (modified)</td><td>Thermo Fisher Scientific</td><td>4467084</td><td>Pre-designed assay modified with VIC-dye: AGO1: Hs02320401_cn</td></tr><tr><td>TaqMan Copy Number Reference Assay, human, RNase P</td><td>Thermo Fisher Scientific</td><td>4403326</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Thermo Fisher Scientific</td><td>4326708</td><td>Master mix for Real Time PCR</td></tr><tr><td>QIAamp DNA Mini Kit (50)</td><td>Qiagen</td><td>51304</td><td>DNA extraction kit</td></tr><tr><td>MicroAmp 96-Well Plates</td><td>Thermo Fisher Scientific</td><td>N8010560</td><td>plates for realt time PCR</td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>-</td><td>the user could use also other spectrophotometric methods to quantify DNA</td></tr><tr><td>7500 Fast Real-Time PCR System</td><td>Applied Biosystem</td><td>-</td><td>the user could use also other real time instrument</td></tr><tr><td>CopyCaller Software</td><td>Applied Biosystem</td><td>-</td><td>Software for copy number analysis</td></tr></tbody></table>

serum and plasma copy number detection using real-time pcr

Serum ve Plazma hücre ücretsiz DNA (cfDNA) biyolojik kanser tanısı, prognoz, izleme ve tedavi direnci tahmini için bir bilgilendirici, non-invaziv kaynağı olarak gösterilmiştir. Androjen reseptör (AR) Gen kopya numarası (CN) kazanç varsayımını başlayarak sık olay metastatik kastrasyon direnç prostat kanseri (mCRPC), bu durumda cfDNA bir potansiyel akıllı biyomarker olarak analiz etmek önerdiğimiz.
Biz AR CN 2 farklı gerçek zamanlı PCR deneyleri ve 2 başvuru genler (RNaseP ve önce1) kullanarak cfDNA olarak değerlendirildi. DNA bir miktar olan 60 ng her tahlil birleşim için kullanıldı. AR CN kazanç dijital PCR daha doğru bir yöntemle doğrulandı. CN varyasyon analizi zaten tedavi direnci mCRPC ortamda tahmin için bilgilendirici olmak kanıtlanmıştır, ama aynı zamanda farklı hasta ortamlarda başka amaçlar için yararlı olabilir. CfDNA CN analiz çeşitli avantajları vardır: non-invaziv, hızlı ve gerçekleştirmek kolay ve serum veya plazma malzeme küçük bir birimden başlar.

Toplam cfDNA konsantrasyonu ölçülebilir spectrophotometry için tüm örneklerini analiz, 6,12 ng/µL sayılarının gösterilen tarafından (aralığı: 2,00-23.71 ng/µL) serum örnekleri ve 3.21 ng/µL ortancası (aralığı: 2.31 8,49 ng/µL) plazma numuneleri için. Gerçek zamanlı PCR deneyler tarafından Toplam 115 örnekleri analiz ettik.
Test duyarlılık ARve DNA için yüksek veya düşük kazancı da Kal...

Watch this Scientific Journal Video about Serum and Plasma Copy Number Detection Using Real-time PCR at JoVE.com

Serum and Plasma Copy Number Detection Using Real-time PCR

Bu el yazması kopya numarası varyasyon analizi serum veya plazma DNA gerçek zamanlı PCR yaklaşımı kullanarak gerçekleştirilen açıklar. Bu yöntem için tahmin kastrasyon dayanıklı prostat kanseri hastalarda ilaç direnci uygundur, ama aynı zamanda diğer hastalıklar için bilgilendirici olabilir.

Serum ve plazma kopya sayı algılama gerçek zamanlı PCR kullanarak

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

serum-and-plasma-copy-number-detection-using-real-time-pcr

Research

JoVE Journal

Cancer Research

11.3K Görüntüleme.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. Bu el yazması kopya numarası varyasyon analizi serum veya plazma DNA gerçek zamanlı PCR yaklaşımı kullanarak gerçekleştirilen açıklar. Bu yöntem için tahmin kastrasyon dayanıklı prostat kanseri hastalarda ilaç direnci uygundur, ama aynı zamanda diğer hastalıklar için bilgilendirici olabilir.

Video: Serum and Plasma Copy Number Detection Using Real-time PCR

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Standart Klinik Tahliller Algılama Sınırı Aşağıda Viral Yükler HIV enfekte kişilerde kuvvetlendirme ve HIV-1 RNA miktarının

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

İmmünoloji ve Enfeksiyon

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

qKAT: nicel Semi-automated yazmaktan katil hücreli immünglobulin benzeri reseptör gen

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Genetik

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Hasta Biofluidinde tümör Ilişkili sirkülasyon DNA 'Sı tespiti ve Izlenmesi

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Kanser Araştırmaları

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T-hücre reseptörleri Eksizyon Circles Eşzamanlı Niceleme (TREC) ve K-silme Rekombinasyon Eksizyon Circles (KRECs) Gerçek zamanlı PCR ile

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

MicroRNA Niceleme DNA bağlayıcı Boya Kimya ve Damlacık Dijital PCR kullanarak Sirkülasyon

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Biyomühendislik

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-tabanlı Real-time PCR microRNA Kantitatif Ölçüm Yaklaşımları

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Biyoloji

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

Dijital Damlacık Polimeraz Zincir Reaksiyonu Kullanarak Tek Hücreli Mitokondriyal DNA Kopya Sayısını ve Heteroplazmiyi Ölçme

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

Tıp

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription 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 approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

Cynomolgus maymunlar, nicel gerçek zamanlı ters transkripsiyon PCR kullanarak plazma mikroRNA düzeylerinin mutlak miktar

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 ...

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is carried out either using fluorescent dyes or sequence-specific probes linked to a fluorophore. The fluorescent dyes used in this case can specifically bind to double-stranded DNA and exhibit fluorescence only when bound to DNA. Fluorescence is measured at the end of each PCR cycle after the synthesis of the complementary strand. In the case of fluorophore-labeled probes, fluorescence is exhibited when the fluorophore is cleaved from the probe during the synthesis of the complementary strand. Fluorescence exhibited by these molecules is then detected by photodetectors that convert the fluorescence signals to a readable format. Real-time RT-PCR requires a specialized PCR machine equipped with a detector to enable real-time quantification, as a traditional PCR machine is not equipped for such analysis.
The output can be analyzed in a number of ways. One way is to count the number of cycles required for the fluorescence to reach detectable levels. The threshold cycle, denoted by Ct, is when the fluorescence is detected over and above the background noise. The greater the quantity of the target in the starting material, the faster the significant increase in fluorescence will appear, resulting in a lower Ct. The Ct value finds application in the downstream quantification or detection of the presence or absence of the target sequence. Additionally, comparing the Ct values of samples of unknown concentration with a series of Ct values obtained from standard concentration can determine the amount of template in an unknown reaction. This is known as absolute quantification and can also be carried out by comparing the fluorescence to a standard curve prepared using known DNA concentrations.

Real-time RT-PCR: Principle; Quantifying Gene Expression

Gerçek Zamanlı RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target ...