ああ、我々の話が中断されたからだね。阿久悠がああいもんで。でもちょうどよかった。
僕はThe relative expression levels of pluripotency-associated genes to Gnb2l1 were indicated with standard deviation. の
意味が分かんなかったんだよ。Gnb2l1が何なのか教えてくれないかな。
Klf4 expression was detected in all samples, which may reflect its expression in liver cells, and thus serves as a positive control in this assay.
すべてのサンプルでKlf4の安定した発現があり、適正な内部標準遺伝子と判断しているようです。
(a) Q-PCR analysis of the low-pH treated liver cells cultured for 7 days. Liver cells were prepared from 7-day old GOF mice and treated with either ATP or HCl, or without stressor. RNA samples were prepared from all cells in the wells at day 7 of culture and the relative expression levels of Gfp (derived from GOF Tg) and Oct3/4 (derived from the endogenous Pou5f1 allele) to Gapdh were indicated with standard deviation. The expression levels in control ES cells carrying CAG-GFP Tg were set at 1.0. (b) Q-PCR analysis of the single cell aggregates derived from the ATP-treated or non-treated liver cells cultured for seven days. The liver cells were prepared from 4-days old of C57BL6/129 mice and the single cell aggregates were separately treated for quantification of gene expression. The relative expression levels of pluripotency-associated genes to Gnb2l1 were indicated with standard deviation. The expression levels in 10 control ES cells were set at 1.0. (c) Frequency of cell aggregates showing the levels of Oct3/4 expression comparable to ES cells. The relative expression levels of Oct3/4 in single cell aggregates derived from liver cells were measured as b and the frequency of the cell aggregates with the levels of Oct3/4 expression over 0.001 of relative expression to ES cells is indicated.
To quantify the levels of mRNA transcripts, total RNA was prepared by TRIzol® (Life Technologies). cDNA were synthesized from 1 μg of total RNA using SuperScript® III (Life Technologies), and quantified by real-time PCR using a CFX384 system (BioRad). All samples were tested in triplicate, and the mean relative amounts of each transcript were calculated by normalization to an endogenous control Gapdh.
Each cell aggregate was washed with PBS and transferred in 2 μl of PBS into 8 μl RealTime ready Cell Lysis Buffer (Roche) supplied with NP-40, RNAsin and RNase inhibitor. Then 3 μl of cell lysis solution was mixed with 1.5 μl of DNaseI solution (0.2 U/μl) to degradate genomic DNA followed by addition of 1.5 μl of 8 mM EDTA solution to stop the reaction. For reverse transcription of RNA, 3 μl of pre-mixture of SuperScript® VILO reverse transcriptase (Life Technologies) was added into 6 μl of DNaseI-treated cell lysate and incubated at 42 °C for 1 hour. The reverse-transcribed product was pre-amplified with Plutinum multiplex PCR master mix using pooled primer mixture using the reaction cycle (95 °C for 30 sec; 60 °C for 90 sec; 72 °C for 60 sec) for 14 cycles. The mixture was treated with Exonuclease I to remove the primers for pre-amplification, and quantitative PCR was performed with the primer pairs specific for each gene using Quantitest SYBR Green PCR mix (Qiagen) in BioRad CFX384 Real-Time System (Bio-Rad). All samples were tested in triplicate, and the mean relative amounts of each transcript were calculated by normalization to an endogenous control Gapdh or Gnb2l1.
All samples were tested in triplicate, and the mean relative amounts of each transcript were calculated by normalization to an endogenous control Gapdh or Gnb2l1.
