Welcome to GeneProof website!

It is great to see you here. To see all contents of the website, please register or restore your access.
We believe you will come back gladly.

GeneProof Team

 

User account
Forgot your password?
Searching

Nucleic Acid Extractions

Weak/missing signal for amplification of IC in HEX channel (HCV PCR Kit) when using manual extraction of RNA?

Possible causes 

  • Higher concentration of RNAses in clinical samples
  • Mistake during addition of IC
  • Incorrect storage conditions for IC

Problem solution

  • Add lysis buffer first, vortex with sample and then add IC
  • Control correct volume of IC which is calculated according elution volume (see IFU)
  • Check the storage conditions and expiration dates Internal Control (indicated on the tube).
Nucleic Acid Extractions Real Time PCR

Was the answer helpful?

YES NO Contact our technical support.

Weak/missing signal for amplification of IC in HEX channel (HCV PCR Kit) when using croBEE NA extraction with a cartridge 201?

Possible causes

  • Higher concentration of RNAses in clinical samples
  • Error during the addition of IC
  • Incorrect storage conditions for IC

Problem solution

  • Add Proteinase K to a sample, vortex it and then incubate for 5 min. at RT. Continue with croBEE NA extraction.
  • Control the correct volume of IC which is calculated according to the elution volume (see IFU)
  • Check the storage conditions and expiration dates IC (indicated on the tube).
Nucleic Acid Extractions Real Time PCR

Was the answer helpful?

YES NO Contact our technical support.

Weak/missing signal for amplification of IC in HEX channel when using manual extraction. The lysis buffer was mixed with Internal Control and stored in a refrigerator.

Possible causes

  • Instability of IC in lysis buffer
  • Problem with solubility of IC in thy lysis buffer

Problem solution

 

????????????

Nucleic Acid Extractions Real Time PCR

Was the answer helpful?

YES NO Contact our technical support.

We do not pass the EQA panel correctly. Our results have low quantity in comparison with the expected results.

Possible causes

  • Unprecise amplification of the calibration curve

Problem with the quality of NA isolation

Problem solution

  • Check the quality of amplification for all four calibrators (if the R2 coefficient is lower than 0.99, repeat PCR detection with the whole calibration set again)
  • Control the quality of nucleic acid extraction.  Check the correct amplification of Internal Control and if the signal for IC in HEX channel is low, check and repeat extraction/PCR detection of samples
Nucleic Acid Extractions Evaluation of Results

Was the answer helpful?

YES NO Contact our technical support.

We have tested a HPVS PCR KIT with standard clinical samples. All results were negative in all detected fluorescence channels. Positive control works properly in all channel.

Possible causes

  • Problem with the correct sampling (device/tool for the collection of samples for HPV test)
  • Problem with the quality of extraction of nucleic acids

Problem solution

  • HPVS PCR kit contain a system for sample quality control (based on housekeeping gene detection). Check the system for sample collection and the recommended sampling for HPV detection.
  • Control the quality of NA extraction. Check the correct amplification of Internal Control and if the signal for IC in HEX channel is low, check and repeat the extraction/PCR detection of samples.
Sampling, transport and storage of samples Nucleic Acid Extractions

Was the answer helpful?

YES NO Contact our technical support.

All results were negative in both channels (FAM/HEX) when using FII/FV/FXIII/MTHFR PCR detection Kit

Possible causes

  • Incorrect sampling or clinical material for detection of human DNA
  • Problem with quality of NA extraction
  • Problem with an incorrect setup in PCR device

Problem solution

  • For the detection of human DNA it is necessary to use whole blood collected in EDTA. Check the clinical material for the extraction.
  • Control the quality of NA extraction. Try to check the concentration of human DNA by spectrophotometer. If there is low/zero concentration of human DNA change/repeat the NA extraction.
  • Check if the PCR device is validated for this setup correctly (PCR profile, fluorescence channels …)
Sampling, transport and storage of samples Nucleic Acid Extractions Real Time PCR

Was the answer helpful?

YES NO Contact our technical support.

Problem with the extraction of nucleic acid from frozen whole blood.

Possible causes

  • Degradation of frozen whole blood during storage
  • Bad sampling format for whole blood collection
  • Extraction method is not validated for the isolation of NA from whole blood

Problem solution

  • For the examination of whole blood it is not recommended to store the sample in a freezer. in a and proceed the extraction in 24 hours.
  • It is necessary to collect whole blood for PCR in EDTA.
  • Check if the extraction method is validated for the isolation of NA from whole blood.
Sampling, transport and storage of samples Nucleic Acid Extractions

Was the answer helpful?

YES NO Contact our technical support.

Absence of signal in hex fluorophore fluorescence channel. Detection in fam fluorophore fluorescence channel works properly.

Possible causes

  • Incorrect choice of HEX fluorescence channel
  • PCR device failure
  • Incorrect storage conditions for IC

Problem solution

  • Select HEX (VIC, JOE) fluorophore fluorescence channel for the detection of internal standard in the correct PCR step according to the Package insert instructions.
  • Perform the test with another PCR kit on the same PCR device. If the problem repeats, consider validation of the instrument.
  • Check the storage conditions and expiration dates of Internal Control (indicated on the tube).
Nucleic Acid Extractions Real Time PCR

Was the answer helpful?

YES NO Contact our technical support.

Absence of signal in HEX fluorophore fluorescence channel of the negative control in ISEX version of the PCR kit. Positive control is amplified properly.

Possible cause

  • When using SEX version the Internal Standard must be added to analysed samples before the Nucleic Acid Extraction. The PCR Negative Control sample does not undergo the isolation process therefore the Internal Standard is not present in the PCR reaction mix and consequently cannot be amplified.

Problem solution

  • This result is normal when using ISEX version of PCR kit and is valid for interpretation.
Nucleic Acid Extractions

Was the answer helpful?

YES NO Contact our technical support.

All extracted samples are positive in FAM fluorophore fluorescence channel. Negative PCR control is negative.

Possible causes

  • Contamination of samples during the extraction process.

Problem solution

  • Changevials, filtered pipette tips and plastics in the extraction box.
  • Clean the extraction box with DNA-OFF solution.
  • Include extraction negative control - an additional control of the cross-contamination during the extraction (sterile water with the same amount of internal standard as in samples).
  • Repeat the extraction of the samples.
Nucleic Acid Extractions Real Time PCR

Was the answer helpful?

YES NO Contact our technical support.

Do you need more information?

Contact form
Choose file
Choose file

Your personal data will be processed according to the privacy policy .