This kit is for preparing genes derived from immune cells, including T cells, from samples containing white blood cells. This kit allows for easy extraction and purification of mRNA from white blood cells in a sample, and synthesis of cDNA by reverse transcription of the purified mRNA. The synthesized cDNA can be used to evaluate relative changes in gene expression by quantitative PCR. This kit is suitable for performing the Ex vivo Activation of Gene in Leukocyte (EAGL) method, a cellular immunity evaluation method based on gene detection. Two uniquely designed plates, the "Leukocyte Isolation Plate" and the "mRNA Capture Plate," achieve high reproducibility and throughput.
| Applications |
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|---|---|
| Work time | 180 minutes + antigen stimulation time |
| Sample Type |
|
| Final product | 30µl cDNA |
| Detection target | Control genes (human, mouse)
|
| Measuring Equipment |
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| application |
|
| Component | Size |
|---|---|
| PT Buffer | 15mL x 1 bottle |
| Leukocyte Isolation Plate (Filter Plate) | 1 sheet |
| Deep Well Plate | 1 sheet |
| Proteinase K | 30 µL x 1 |
| TCEP | 300 µL x 1 |
| Lysis Buffer | 6 mL x 1 bottle |
| mRNA Capture Plate | 1 sheet |
| Wash Buffer A | 30mL x 1 bottle |
| Wash Buffer B | 50 mL x 1 bottle |
| Aluminum Seal | 1 sheet |
| M-MLV Reverse Transcriptase | 40 µL x 1 |
| RNase Inhibitor | 10 µL x 1 |
| RT Buffer | 1.5 mL x 2 |
| Component | Size |
|---|---|
|
Antigen Coated Plate for SARS-CoV-2 *1 |
1 sheet (24 tests) |
*1 Overlapping peptides (15 amino acids, 11 overlaps) derived from the SARS-CoV-2 spike protein are applied as antigens. One test consists of four wells: a blank, two antigens, and a positive control.
| Components | Size |
|---|---|
| Primer Mix ※2 | 100 µL x 1 |
| PCR Enzyme Mix | 500 µL x 1 |
*2 The part number and included Primer Mix vary depending on the gene of interest.
Product data
Gene extraction performance
- Relationship between the amount of each sample (human blood, human PBMC, mouse blood, mouse spleen) added and the expression level of the control gene -
Figure 1. Human blood and mouse blood were 50 mL, and human PBMC and mouse spleen were serially diluted in two-fold increments starting from 5 x 105 cells , and mRNA from leukocytes was extracted/cDNA was synthesized using LeukoComplete™. Housekeeping genes were measured by real-time PCR using the synthesized cDNA as a template. For all sample types, genes were extracted with high linearity (R2 > 0.9) using LeukoComplete™.
Examples of target genes
- Analysis of the expression changes of immune-related genes (IFNG, IL2, IL4, GM-CSF) according to the time of antigen stimulation -
Figure 2. 100 µL of fresh human blood was mixed with CEF peptide pool or PBS, and after incubation at 37°C for 0 to 24 hours, the blood was frozen and stored. After thawing the blood, genes were extracted using LeukoComplete™ and the expression changes of various genes were calculated (each colored line represents the results of a different donor (n=4)). log 2 FC = - (Ct value of target gene [with antigen stimulation] - Ct value of control gene [with antigen stimulation]) - (Ct value of target gene [without antigen stimulation] - Ct value of control gene [without antigen stimulation])
