24小时热门版块排行榜

返回列表

xiaomcss

新虫 (初入文坛)

应助: 0 (幼儿园)
金币: 21
帖子: 5
在线: 7.3小时
虫号: 4016988
注册: 2015-08-11
性别: MM
专业: 污染控制化学

[求助] 求大神帮忙翻译一下下面论文里面的MATERIALS AND METHODS部分

本从刚刚注册了账户，没有太多金币，以后定当重谢！！不用全部翻译，求大神把里面实验步骤，所以材料、设计等翻译出来就好，我想要重复一下这个实验，谢谢谢谢！！

论文题目 Real-time quantification of microRNAs by stem–loop RT–PCR

MATERIALS AND METHODS

Targets, primers and probes (Supplementary Data)

Seventeen miRNA genes were selected from the Sanger Center miRNA Registry at http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml. All TaqMan miRNA assays are available through Applied Biosystems (P/N: 4365409). Standard TaqMan® assays for pri-miRNA precursors, pri-let-7a-3 and pri-miR-26b and pre-miRNA precursor pre-miR-30a were designed using PrimerExpress® software (Applied Biosystems, Foster City, CA). All sequences are available in the section of the Supplementary Data. Synthetic miRNA oligonucleotides were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

Tissue RNA samples, cells, cell lysates and total RNA preparation

Mouse total RNA samples from brain, heart, liver, lung, thymus, ovary and embryo at day 10–12 were purchased from Ambion (P/N: 7810, 7812, 7814, 7816, 7818, 7824, 7826 and 7968). Ambion's mouse total RNAs are derived from Swiss Webster mice. All RNA samples were normalized based on the TaqMan® Gene Expression Assays for human or mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous controls (P/N: 4310884E and 4352339E, Applied Biosystems).

Two cell lines, HepG2 and OP9, were cultured using Gibco MEM (P/N: 12492–021, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (P/N: SH30070.01, HyClone, Logan, UT). Trypsinized cells were counted with a hemocytometer. Approximately 2.8 × 106 suspended cells were pelleted by centrifugation (Allegra 6, Beckman Coulter, Fullerton, CA) at 1500 r.p.m. for 5 min, washed with 1 ml Dulbecco's phosphate-buffered saline (PBS) without MgCl2 and CaCl2 (P/N: 14190078, Invitrogen, Carlsbad, CA). The cell pellets were re-suspended in 140 μl PBS and processed with three different sample preparation methods. With the first method, a 50 μl sample (106 cells) was mixed with an equal amount of Nucleic Acid Purification Lysis Solution (P/N: 4305895; Applied Biosystems) by pipetting up and down 10 times, and then spun briefly. The lysate was diluted 1/10 with 1 U/μl RNase inhibitor solution (P/N: N8080119; Applied Biosystems) before adding the solution to an RT reaction. In the second method, a 50 μl sample (106 cells) was used to purify total RNA using the mirVana™ miRNA Isolation Kit (P/N: 1560, Ambion, Austin, TX) according to the manufacturer's protocol. Purified total RNA was eluted in 100 μl of elution buffer. The third method involved diluting cells 1/2 with 1× PBS, heating at 95°C for 5 min, and immediately chilling on ice before aliquotting directly into RT reactions.

miRNA detection using mirVana™ miRNA detection kit

Solution hybridization-based miRNA analysis was carried out using the mirVana™ miRNA Detection Kit (Cat. #: 1552, Ambion) according to the manufacturer's protocol. RNA probes were synthesized by IDT. The radioisotope labeled RNA fragments were detected and quantitated with a Cyclone Storage Phosphor System (PerkinElmer, Boston, MA).

Reverse transcriptase reactions

Reverse transcriptase reactions contained RNA samples including purified total RNA, cell lysate, or heat-treated cells, 50 nM stem–loop RT primer (P/N: 4365386 and 4365387, Applied Biosystems), 1× RT buffer (P/N: 4319981, Applied Biosystems), 0.25 mM each of dNTPs, 3.33 U/μl MultiScribe reverse transcriptase (P/N: 4319983, Applied Biosystems) and 0.25 U/μl RNase inhibitor (P/N: N8080119; Applied Biosystems). The 7.5 μl reactions were incubated in an Applied Biosystems 9700 Thermocycler in a 96- or 384-well plate for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C and then held at 4°C. All Reverse transcriptase reactions, including no-template controls and RT minus controls, were run in duplicate.

PCR

Real-time PCR was performed using a standard TaqMan® PCR kit protocol on an Applied Biosystems 7900HT Sequence Detection System (P/N: 4329002, Applied Biosystems). The 10 μl PCR included 0.67 μl RT product, 1× TaqMan® Universal PCR Master Mix (P/N: 4324018, Applied Biosystems), 0.2 μM TaqMan® probe, 1.5 μM forward primer and 0.7 μM reverse primer. The reactions were incubated in a 384-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. All reactions were run in triplicate. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. TaqMan® CT values were converted into absolute copy numbers using a standard curve from synthetic lin-4 miRNA.

The method for real-time quantification of pri-miRNA precursors, let-7a-3 and miR-26b, and pre-miRNA precursor miR-30a was described elsewhere (34).