| Questions |
Answers |
| a. |
How should I store the kit? |
The PG5701 Detection Kit should always be kept at -20°C in the dark and thawed on the ice while using. |
| b. |
Can I use blood collection tubes containing Heparin as anticoagulant? ? |
It is highly recommended to use blood collection tubes containing either sodium citrate or EDTA as the anticoagulants. Heparin may have an inhibitory effect on PCR. |
| c. |
Are there preferable DNA extraction kits for extraction gDNA? |
Qiagen QIAamp® DNA Blood Mini Kit is recommended. |
| d. |
What is the required quality of gDNA? |
The OD260/280 ratio of gDNA should be between 1.7 and 2.0. |
| e. |
What is the appropriate amount of DNA in the reaction? |
The appropriate amount of gDNA should be between 25-100 ng/reaction. Inappropriate amount of gDNA, over 100 or less than 25 ng/rxn, may cause the Ct value of internal control over 27. |
| f. |
How many reactions should be conducted for each sample? |
Two independent PCR reactions should be conducted simultaneously for each DNA sample. One is for HLA-B*5701 detection and the other one is for internal control gene detection. |
| g. |
What dyes does the Kit use? |
PG5701 Detection Kit uses SYBR® Green I dye.
You can select channel SYBR or FAM (same detection wave length) as your Detection fluorescence.
|
| h. |
What are the Quencher and Reference dye setting I need to use on my instrument? |
The quencher setting should be set to ‘None’.
If your instrument needs to select reference dye, please select Rox dye. |
| i. |
How should I set the threshold? |
The threshold value setting depends on the quantitative PCR instruments you use. Please refer to section 4.4 of PG5701 Detection Kit package insert for the setting of threshold value on different models of real-time PCR system. |
| j. |
How do I distinguish HLA-B*5701 positives from negatives? |
Samples are identified as HLA-B*5701 positives when IC Ct ≤ 27 and the ㅿCt ≤ 7. When the ㅿCt > 7, it means HLA-B*5701 negatives. |
| k. |
Do I need to set different thresholds for getting Ct values of Genotype and Internal control? |
One threshold is enough for both targets because they have very similar amplification slopes. |
| l. |
If Genotype Detection shows “Undetermined”, how do I explain the results? |
If you conduct the tests correctly, then “undetermined” means negative signal. Ct values of Genotype Detection do not always exist unless particular alleles sequence are similar to HLA-B*5701. When the ㅿCt ≤ 7, it means HLA-B*5701 positives. |
| m. |
Does internal control always have ct value? |
Internal control is similar to a housekeeping gene. Therefore, it should have signal in each specimen and we can monitor the amount and quality of gDNA depending on its data. |
| n. |
Do I need to duplicate my tests? |
Based on the description in package insert, duplication is not always needed. It needs to be conducted according to the QC guidance of each lab. |
| o. |
Why does the NTC show amplification signal? |
NTC is a control without DNA sample. Ct value shown represents an artificial mistake and has DNA contamination(s) in samples. |
| p. |
How do I set up the software program of the quantitative PCR instrument? |
Pharmigene provides program setting instructions for a few quantitative PCR detection systems. Please do not hesitate to contact us for the further information if needed. |
| q. |
How do I contact the Technical Support department of PharmiGene? |
Please call +886-2-26959800 or email service@pharmigene.com to request a PGI’s service. |
