研究思路
研究方法
實(shí)驗(yàn)樣本的選取
選擇腫瘤細(xì)胞豐富的區(qū)域凄硼，用手動(dòng)打孔機(jī)鉆取組織樣本孕讳，用數(shù)碼相機(jī)拍照匠楚，然后用ImageJ軟件測量出組織體積。
提取試劑盒的選擇
一共14個(gè)試劑盒厂财，包含了16種提取DNA和RNA的方法芋簿，其中RecAll和AllPrep既可以提取RNA又可以提取DNA。
核酸的提取及定量
樣本前處理：從4塊歸檔的FFPE組織石蠟塊中鉆取了120個(gè)樣璃饱，共計(jì)37.2mm3与斤。用二甲苯室溫脫蠟5分鐘兩次，無水乙醇室溫復(fù)水5分鐘兩次，然后使用勻漿機(jī)勻漿幽告。
核酸提让佛小：每份樣本0.68mm3,按照各個(gè)試劑盒說明書提取，每個(gè)試劑盒三重復(fù)冗锁。其中RecAll和AllPrep試劑盒提取時(shí)齐唆，在勻漿后的組織中添加蛋白酶K，然后取上清提取RNA冻河，沉淀提取DNA箍邮。
核酸產(chǎn)量及純度檢測：使用dsDNA HS和RNA BR在Qubit3.0上分別對(duì)DNA及RNA定量；使用分光光度計(jì)測定A260/280和A260/230判定DNA及RNA純度叨叙。
Limitations of this study
This study has some notable limitations. Kit performance was tested on cores but not on tissue sections, which may be better suited for certain applications. Nevertheless, cores are a popular and efficient method of selecting tissue of interest for molecular assays. In addition, the head to-head kit comparison implemented was based on relatively recent tissue samples. Older samples were evaluated with the AllPrep kit but not with any of the other kits.
研究結(jié)果
1. 核酸產(chǎn)量和完整性
By spectrophotometric assessment, all of the RNA extraction kits produced A260/280 ratios close to 2.0, consistent with highly “pure” samples. In contrast, with the exception of the RNeasy kit, all of the RNA extraction kits produced A260/230 ratios that indicated significant impurities (Table 2). Similarly for DNA, the A260/280 ratios were near or above 1.8 for all kits. In contrast, the A260/230 ratios for the RecAll, AllPrep, QIAamp, and DNeasy kits indicated potential organic contaminants in the eluent (Table 2). Contaminants such as EDTA, phenol, heme, and carbohydrates all have absorbances near 230 nm. Given that these contaminants can inhibit downstream applications, we undertook a series of assays to quantify their inhibitory effect.
2. PCR抑制劑效果檢測
The inhibition assay was designed to quantify the impact of any inhibitors in eluted RNA and DNA samples on downstream applications. Extracted RNA and DNA were spiked into a non-target PCR reaction, and the observed delay in the Cq value of the RNA- or DNA-spiked reaction relative to that of the control (water-spiked) was interpreted as the extent of the inhibitory effect. 單因素方差分析結(jié)果顯示锭弊，8個(gè)RNA提取試劑盒的Cq值和Control的Cq值基本在同一水平，表明RNA純度較高擂错；6個(gè)DNA提取試劑盒中有5個(gè)試劑盒的Cq值和Control的Cq值基本在同一水平味滞，但AllPrep和對(duì)照組有明顯差異。
AllPrep試劑盒PCR抑制劑補(bǔ)充實(shí)驗(yàn)
額外選取了前列腺癌和乳腺癌樣本進(jìn)行抑制實(shí)驗(yàn)钮呀，發(fā)現(xiàn)抑制現(xiàn)象只在前列腺癌樣本中發(fā)生剑鞍。前列腺癌樣本DNA分別稀釋十倍和二十倍再進(jìn)行實(shí)驗(yàn)，在稀釋十倍時(shí)抑制情況有明顯改觀爽醋。
結(jié)論：
測試的試劑盒中蚁署，AllPrep提取的DNA中存在PCR抑制劑（僅在前列腺癌樣本中），但可以通過稀釋消除抑制效應(yīng)蚂四；其余試劑盒提取的DNA和RNA與對(duì)照相比光戈，沒有明顯的抑制效應(yīng)。
3. 核酸片段大小的評(píng)估
方案1：RNA樣本使用RNA 6000 Pico 試劑盒在2100生物分析儀上檢測遂赠，用大于200bp堿基對(duì)百分比（DV200*）表示久妆。
結(jié)果見表2，相同樣本用不同試劑盒抽提獲得的DV200值相差很大跷睦。
*DV200值表示的是片段大于200bp的相對(duì)數(shù)量镇饺，而不是絕對(duì)數(shù)量，并不能用于反映進(jìn)入PCR反應(yīng)的核酸的性能送讲。
方案2：評(píng)估RNA片段分布時(shí)先進(jìn)行逆轉(zhuǎn)錄，然后用擴(kuò)增子大小從92bp到386bp（大約以50bp大小遞增）的6對(duì)堿基對(duì)和cDNA進(jìn)行PCR擴(kuò)增惋啃；評(píng)估DNA片段分布時(shí)用擴(kuò)增子大小從102bp到300bp（大約以65bp大小遞增）的4對(duì)堿基對(duì)和DNA進(jìn)行PCR擴(kuò)增哼鬓；擴(kuò)增后產(chǎn)物用3%凝膠電泳質(zhì)檢。
In the current study, no clear correlation was observed in the fragment size distribution as determined by end-point PCR versus Bioanalyzer analysis. It is noteworthy, however, that extensive fragmentation of both RNA and DNA, which is typical for FFPE samples, made the interpretation of the Bioanalyzer electropherogram unreliable(S1 Fig).
Based on yield and purity, five DNA protocols (AllPrep, RecAll, DNeasy, QIAamp, and GenJet) and four RNA protocols (AllPrep, RecAll, PuLink, and RNeasy) were prioritised for further quality assessment. The prioritised kits were further evaluated using NanoString, RT-qPCR expression, and MS-PCR assays.
4. NanoString和實(shí)時(shí)熒光定量法對(duì)mRNA 檢測
The NanoString platform has previously been used for direct, digital quantitation of specific mRNA transcripts through hybridization to two sequence-specific, color-coded probes . As this technology is strictly hybridization-based, it avoids the use of reverse transcription and amplification, and thereby eliminates potential amplification bias common to PCR.
Pairwise comparisons between RNA extracted from the cell line and each of the four selected kits showed no significant difference in total signal counts (Fig 2A and S2 Fig).
By RT-qPCR, RNA from all four prioritized RNA protocols were successfully used to amplify the three house-keeping genes tested (PGK1, KRT8 and HPRT1), showing the overall compatibility of these protocols with downstream gene expression assays. Of these, the AllPrep protocol yielded significantly higher Cq values for all three genes (two-way ANOVA, p < 0.0001; Fig 2B). 原因是RNA中有PCR抑制劑边灭。
5. 甲基化特異性PCR檢測DNA
We targeted three genes known to be hypermethylated in prostate cancer, namely GSTP1, ABCB1, and RASSF1, along with the Alu repeat sequences as a positive control. DNA from the AllPrep, DNeasy, and GenJet kits resulted in similar Cq values (Fig 2C). Compared to the other kits, and using equivalent amount of DNA input, RecAll and QIAamp had significantly higher Cq values for each of the three gene targets (two-way ANOVA, p < 0.05).
These results indicated that AllPrep DNA was most compatible with the methylation-specific PCR protocol used in this study.
**6. AllPrep提取方案重現(xiàn)性
Inter-laboratory validation of a molecular protocol or assay is performed by molecular pathology laboratories to ensure its reproducibility and accuracy, which are critical components of competent patient care. We aimed to identify the optimal RNA and DNA extraction protocols and then determine whether they are reproducible across multiple laboratories.
We placed a high priority on the metrics routinely used in pre-analytical quality control, such as spectrophotometric absorbance and fragment distribution, and selected Qiagen’s AllPrep DNA/RNA FFPE kit as the optimal one for assessing the reproducibility of extraction protocols between three laboratories.
One-way ANOVA analysis did not detect significant differences between laboratories in yield of DNA or RNA (p > 0.05, Fig 3A), or in the absorbance at 280, 260, and 230 nm (results not shown). The results of the MS-PCR (S3C Fig) and NanoString (S3D Fig) assays using nucleic acids extracted independently by the three laboratories were highly correlated (all Pearson’s R2 ≥ 0.94; p < 0.0001).
Thus, the AllPrep kit produced nucleic acids that strongly correlated between the three laboratories in yield, quality, and compatibility with downstream applications.
7. Effect of sample age on yield and downstream molecular applications
The 12 FFPE samples used in the inter-laboratory study on the AllPrep kit ranged in age from 7 to 15 years. Pearson’s correlation analysis of the data failed to detect any correlation between the sample age and the yield (Fig 3B). Likewise, sample age was not significantly correlated with MS-PCR Cq values (Fig 3C) or with NanoString total mRNA counts (Fig 3D, p ≥ 0.05).
樣本保存年份與核酸產(chǎn)量之間的沒有相關(guān)性异希；與 MS-PCR Cq values 或 NanoString total mRNA counts之間也沒有相關(guān)性。
參考資料：
- Patel, P.G. et al. Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores. PLoS One 12, e0179732 (2017).
