Description

SuperPrep® Cell Lysis & RT Kit for qPCR (Code No. SCQ-101) consists of "Lysis Reagents" and "RT Reagents" for synthesis of cDNA templates for real-time PCR assays. "Lysis Reagents" prepares cell lysates containing RNAs that can be used as templates for reverse transcription. "RT Reagents" contains reagents for reverse transcription, optimized for efficient cDNA synthesis from crude lysates. The synthesized cDNA can be applied to real-time PCR directly. This assay system is suitable for high-throughput assays.
SuperPrep® Cell Lysis Kit for qPCR (Code No. SCQ-201) is an option of "Lysis Reagents". The cell lysate prepared by "Lysis Reagents" can be applied to one-step real-time PCR.

Features

  • RNA purification is not necessary.
  • High-quality cDNA can be obtained from cell lysates.
  • Reduction of dispersion on high-throughput assay.
  • Various real-time PCR regents can be applied.

Applications

<SuperPrep® Cell Lysis & RT Kit for qPCR>
cDNA synthesis for real-time PCR from mammalian cultured cells (SCQ-101)
total RNA synthesis for real-time PCR from mammalian cultured cells (SCQ-201)

Storage condition

Store at -20ºC

Components

The reagent includes the following components for 20 reactions (SCQ-101S, SCQ-201S) or 100 reactions (SCQ-101, SCQ-201).
SCQ-101S and SCQ-101 contain two separate packages named "Lysis Reagents" and "RT Reagents", respectively. All reagents should be stored at -20ºC.

Table 1.SuperPrep® Cell Lysis & RT Kit for qPCR (SCQ-101, SCQ-101S)

<Lysis Reagents> SCQ-101 SCQ-101S (SAMPLE)
Lysis Solution 5.5 ml 1.1 ml
gDNA Remover 33 µl 6.6 µl
Stop Solution 1.1 ml 220 µl
<RT Reagents> SCQ-101 SCQ-101S (SAMPLE)
5 x RT Master Mix 860 µl 172 µl
5 x RT Master Mix no-RT control 86 µl 17 µl
Nuclease-free Water 2 × 1.7 ml 680 µl

Table 2.SuperPrep® Cell Lysis Kit for qPCR (SCQ-201, SCQ-201S)

  SCQ-201 SCQ-201S (SAMPLE)
Lysis Solution 5.5 ml 1.1 ml
gDNA Remover 33 µl 6.6 µl
Stop Solution 1.1 ml 220 µl
RNase Inhibitor 1.1 ml 220 µl

Application data

Example 1.High-quality cDNA can be obtained from cell lysates.

The optimized lysis solution efficiently inhibits RNA degradation during treatment. RNA in the lysate is stable on ice for at least 2 h. High-quality cDNA can be synthesized using highly efficient reverse transcriptase "ReverTra Ace®" with low contamination of genomic DNA because of preceding DNase I treatment. The reverse transcriptase is supplied as a master mix reagent containing optimally mixed primers (random and oligo dT) to achieve effective cDNA synthesis.

Comparison of the efficiency with purified RNA

SuperPrep® Cell Lysis & RT Kit for qPCR (Code No SCQ-101) synthesized cDNA using the lysate from 2.5×104 HEK293 cells in a 40 µl reaction. cDNA was synthesized using 66.6 ng of total RNA, corresponding to 2.5×104 HEK293 cells, using a cDNA synthesis kit (ReverTra Ace® qPCR RT Master Mix [Code No. FSQ-201]) in a 40 µl reaction. Fifteen housekeeping genes were then analyzed by SYBR® Green real-time PCR using the synthesized cDNA. High correlation was observed between the two methods. The method using SuperPrep® facilitated the expression analysis by real-time PCR without time-consuming RNA purification steps.


Evalution of efficiency for expression analysis of cells with high RNase activity

Expression analysis using a lysate from U937 cells is difficult because of high RNase activity. In this experiment, the lysates (8 µl) from 1×104, 1×103, 1×102 and 1×101 cells were used. β-actin genes were analyzed by SYBR® Green real-time PCR assay using cDNA synthesized from the lysates in 40 µl reaction. The same experiments were performed using two similar systems (SuperPrep® and another company's product [Company A]) and conventional method using corresponding amounts of RNAs. SuperPrep® showed successful amplification, comparable to the conventional method.

The cells tested by this systems.

Call Name Adherent/
Non-adherent
Species Remarks
1 A431 Adherent H.sapiens epidermoid carcinoma cell line
2 C2C12 Adherent M. musculus myoblast cell line
3 Caco-2 Adherent H.sapiens colon adenocarcinoma cell line
4 CHO-K1 Adherent C. griseus ovary cell line
5 COLO205 Non-adherent H.sapiens colon adenocarcinoma cell line
6 DLD-1 Adherent H.sapiens colon adenocarcinoma cell line
7 HCT-15 Adherent H.sapiens colon adenocarcinoma cell line
8 HDF Adherent H.sapiens primary foreskin fibroblasts (primary cell)
9 HEK293 Adherent H.sapiens embryonic kidney cell line
10 HeLa S3 Adherent H.sapiens cervix carcinoma cell line
11 HepG2 Adherent H.sapiens hepatocellular carcinoma cell line
12 HUVEC Adherent H.sapiens umbilical vein endothelial cells (primary cell)
13 Jurkat Non-adherent H.sapiens T lymphocyte cell line
14 K562 Non-adherent H.sapiens myelogenous leukemia cell line
15 KUSA-A1 Adherent M. musculus bone marrow stromal stem cell line
16 L929 Adherent M. musculus aneuploid fibrosarcoma cell line
17 MCF7 Adherent M. musculus breast adenocarcinoma cell line
18 Neuro2a Adherent M. musculus neuroblastoma cell line
19 NIH-3T3 Adherent M. musculus embryo fibroblast cell line
20 PC12 Adherent R. norvegicus adrenal pheochromocytoma cell line
21 rMSC Adherent R. norvegicus bone marrow stromal stem cells (primary cell)
22 THP-1 Non-adherent H.sapiens acute monocytic leukemia cell line
23 U937 Non-adherent H.sapiens leukemic monocyte lymphoma cell line

Example 2.Stability test of the cell lysates.

cDNA were synthesized from lysates that had been left on ice for 0–24 h after lysing of 4×104 HeLa and U937 cells using SuperPrep®. β-actin genes were detected using TaqMan® real-time PCR assay with THUNDERBIRD® Probe qPCR Mix (Code No. QPS-101). The results were compared with that from the other company’s system (Company A). The results suggest that the RNA in the cell lysates is stable for at least 2 h. Lysates from U937 cells showed higher RNase activity than the other cells and tended to deteriorate in storage over 2 h.

NOTE:

RNase activity depends on the type and number of cells. The cell lysates should be placed on ice after preparation and cDNA should be synthesized immediately after preparing the lysates to minimize RNA degradation.

Example 3.Various real-time PCR reagents can be applied.

The synthesis cDNA can be used in various real-time PCR assay (TaqMan® probe, SYBR® Green etc.).
In addition, the cell lysate can be applied to one-step real-time PCR reagents.

TaqMan® Probe assay using cDNA synthesized from cell lysates prepared by SuperPrep®

cDNAs were synthesized using the cell lysates (8 µl) prepared from 7.5×104, 5×104, 1×104, 1×103, 1x102 and 1×10] HeLa S3 cells by SuperPrep® in 40 µl reaction. β-actin genes were detected by various real-time PCR reagents with TaqMan® real-time PCR assay. Successful amplifications were obtained from all reagents tested and the THUNDERBIRD® Probe qPCR Mix tended to show a better Ct than the other tested methods.

Example 4.Evaluation of the assay variation

HeLa S3 cells were incubated with or without 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h after seeding at 2×104 cells/well in a 96-well culture plate. cDNA were synthesized from the lysates prepared from the cells washed with PBS(-). IL-6, IL-1β and β-actin genes were detected by TaqMan® real-time PCR assay with THUNDERBIRD® Probe qPCR Mix (Code No. QPS-101). After compensation of the Cts of IL-6 and IL-1β by that of β-actin, the ΔΔCt between with or without PMA and Z' factors* were calculated.
Z' factors from SuperPrep® were superior to that from the other system (Company A).

*The Z' factor is a simple statistical parameter that is used to assess the quality of high-throughput screening (HTS) assays. A Z' score of ≥0.5 is generally considered to indicate good quality Z' can be calculated by the following formula.

Z'= 1-3 × [Δ Ct(+) standard deviation + Δ Ct(-) standard deviation]/| Δ Δ Ct|