SuperPrep® II cell Lysis & RT Kit for qPCR

Product#: SCQ-401
$1.00
SCQ-401
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SuperPrep® II cell Lysis & RT Kit for qPCR

Cat. No. SCQ-401
Store -20ºC

Description

SuperPrep® II Cell Lysis & RT Kit for qPCR (Code No. SCQ-401) 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. In the previous version, it was necessary to add Stop Solution after adding Lysis Solution. In SuperPrep® II, cell lysates with lysis solution can be used as templates for cDNA synthesis directly. Moreover, high sensitivity detection is possible from a wide variety of mammalian cells than in the previous version. With this assay system, it is possible to synthesize template cDNA for real-time PCR from cultured cells conveniently and quickly.

SuperPrep® II Cell Lysis Kit for qPCR (Code No. SCQ-501) is an option of "Lysis Reagents". The cell lysate prepared by "Lysis Reagents" can be applied to one-step real-time PCR.
SCQ-401_fig1.png 
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.
Application
DNA synthesis for real-time PCR from mammalian cultured cells(Code No. SCQ-401)
Total RNA preparation for one-step real-time PCR from mammalian cultured cells (Code No. SCQ-501)

Components
The reagent includes the following components for 20 reactions (SCQ-401S) or 100 reactions (SCQ-401, SCQ-501).
SCQ-401S and SCQ-401 contain two separate packages named "Lysis Reagents" and "RT Reagents", respectively. All reagents should be stored at -20°C

Table 1.SuperPrep® II Cell Lysis & RT Kit for qPCR (Code No.SCQ-401, SCQ-401S)
 

<Lysis Reagents>

SCQ-401

SCQ-401S (SAMPLE)

Lysis Solution

6.5 ml

1.3 ml

gDNA Remover

33 µl

6.6 µl

RNase Inhibitor

110 µl

22 µl

<RT Reagents>

   

5 × RT Master Mix

860 µl

172 µl

5 × RT Master Mix no-RT Control

86 µl

17 µl

Nuclease-free Water

1.7 ml × 2

680 µl



Table 2.SuperPrep® II Cell Lysis Kit for qPCR (Code No.SCQ-501)
 

Lysis Solution

6.5 ml

gDNA Remover

33 µl

RNase Inhibitor

110 µl


Applications
Example 1.High-quality cDNA can be obtained from several kind of cell lysates.
The optimized lysis solution efficiently inhibits RNA degradation during treatment. RNA in the lysate is stable on ice for at least 6 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.

Table 1 Cells tested by this system
 
 

Call Name

Adherent
Non-adherent

Species

Remarks

1

HPA

Adherent

H.sapiens

preadipocytes (primary cell)

2

HEK

Adherent

H.sapiens

epidermal keratinocytes (primary cell)

3

HA

Adherent

H.sapiens

astrocytes (primary cell)

4

HDF

Adherent

C. griseus

dermal fibroblasts (primary cell)

5

HBEpC

Adherent

H.sapiens

bronchial epithelial cells (primary cell)

6

HUVEC

Adherent

H.sapiens

umbilical vein endothelial cells (primary cell)

7

HPAEC

Adherent

H.sapiens

pulmonary artery endothelial cells (primary cell)

8

HC

Adherent

H.sapiens

chondrocytes (primary cell)

9

HOb

Adherent

H.sapiens

osteoblasts (primary cell)

10

HSkMC

Adherent

H.sapiens

skeletal muscle cells (primary cell)

11

HAOSMC

Adherent

H.sapiens

Aortic smooth muscle cells (primary cell)

12

HFDPC

Adherent

H.sapiens

hair follicle dermal papilla Cells (primary cell)

13

HeLa S3

Adherent

H.sapiens

cervix carcinoma cell line

14

HepG2

Adherent

H.sapiens

hepatocellular carcinoma cell line

15

Jurkat

Non-adherent

H.sapiens

T lymphocyte cell line

16

K562

Non-adherent

H.sapiens

myelogenous leukemia cell line

17

THP-1

Non-adherent

H.sapiens

acute monocytic leukemia cell line

18

U937

Non-adherent

H.sapiens

leukemic monocyte lymphoma cell line

19

HMNC

Non-adherent

H.sapiens

Mononuclar cells (primary cell)

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.
SCQ-401_example2.png
 
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.
SCQ-401_example3.png
 
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 x [Δ Ct(+) standard deviation + Δ Ct(-) standard deviation]/| Δ Δ Ct|
SCQ-401_example4_1.png 

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