Creative Biolabs offers academic and industrial researchers services on iPSC generation and applications. Now our experienced scientists at Creative Biolabs are able to use our matured qPCR technology for the analysis of iPSC pluripotency gene expression.
First demonstrated by Shinya Yamanaka's lab in 2006, the induced pluripotent stem cells (iPSCs) can be generated from different adult somatic cell types by the introduction of four transcription factors (OCT4, SOX2, KLF4, and MYC). With the pluripotency similar to embryonic stem cells (ESCs), iPSCs have shown great potential in drug discovery, disease modeling, and regenerative medicine research. However, the possible low efficiency in pluripotency induction during reprogramming process is the major limitation for the iPSCs generation. In recent years, a number of molecular markers have been identified to verify the pluripotent status of iPSCs which includes Oct4, Nanog, Sox2, Klf4, Ecat, Eras, Fbx15, and Rex1. The process to verify the pluripotency of iPSCs according to well-established markers would be much helpful for the downstream proliferation and differentiation.
A quantitative polymerase chain reaction (qPCR), also known as real-time polymerase chain reaction (Real-Time PCR) is a commonly used technology of molecular biology based on the polymerase chain reaction (PCR). The amplification of a targeted DNA molecule can be monitored in real-time during the PCR process rather than the end in conventional PCR. In general, there are two methods for the detection of PCR products in qPCR. One is that intercalation of non-specific fluorescent dyes with all the double-stranded DNA. The other is the sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which can be detected only after hybridization of the probe with its complementary sequence.
Fig.1 RT-qPCR analysis of general and naïve pluripotency markers in various reset cell cultures.
Since pluripotency modification is demonstrated via different protein synthesis and this originates from gene expression result change. As the source of gene expression alteration, mRNA dynamics is the basis and target in discovering biochemical configuration. Since mRNA is single-strained and can not be assessed via quantitative polymerase chain reaction, it is essential in firstly transferring single-strained mRNA into double-strained complementary DNA (cDNA) in a reaction catalyzed by the enzyme reverse transcriptase. The reverse transcriptasing process along with following polymerase chain reaction form the complete process called rtPCR.
In order to analyze the pluripotent status of iPSCs, we performed rtPCR for the detection of expressed pluripotency gene markers. Now we are able to use both the human iPSC qPCR kit and mouse iPSC qPCR kit to confirm the iPSC with well-established pluripotency markers.
Mouse Kit includes: NANOG, OCT4, SOX2, TBX3, ESRRB, Tcl1, Actin (positive control).
Human Kit includes: NANOG, OCT4, SOX2, UTF1, hTERT, ZFP42(REX1), DEMT3B, G3PDH (housekeeping gene).
With amplification plots of qPCR as raw data available (Figure 2), it is convenient to calculate gene expression level in quantities and compare with cells in different groups of different pluripotency status.
Fig. 2RT-qPCR amplification plot of naïve (blue) and primed (green) embryonic stem cell and induced pluripotent stem cells (red) in pluripotent marker gene OCT-4 expression dynamics.
Creative Biolabs is glad to provide the qPCR and rtPCR analysis for iPSC pluripotency gene markers in custom pathways. Besides，corresponding kits for RNA isolation & purification, RNA transverse transcriptasing and DNA polymerase chain reaction are available for customers' customized operation. Based on our extensive experience, we have the capability to enable you to free up your time for core work and project. If you are interested in our services, please feel free to contact us for more details.
Our qPCR service offers a robust and reliable method for analyzing gene expression levels, making it an instrumental technique for detecting pluripotency markers in iPSCs. Here are the key components of our service.
Below are the findings presented in the article related to qPCR analysis for pluripotency markers for iPSC.
Valentina, et al. characterized iPSC clones comparatively using morphological appearance, alkaline phosphatase (AP), immunocytochemistry, protein blotting, and RT-qPCR analyses in order to test whether changes in reprogramming capacity affect the pluripotent properties of the generated iPSC clones.
The results showed that the expression levels of endogenous pluripotent genes (OCT4, SOX2 and NANOG) were comparable in all characterized clones as well as in hESC and hiPSC cell lines. In contrast, fibroblasts used as negative controls did not show any expression of these genes. This data suggests that all generated iPSC clones have the same morphological appearance, biochemical activity, and known pluripotent gene expression, regardless of their phenotype.
Fig. 3 RT-qPCR analysis of pluripotent genes expressed by the generated iPSCs.2
References
For Research Use Only. Not For Clinical Use.
