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New Method for Rapid Isolation of RNA Directly from Ahlstrom-Munksjö GenSaver™ Cards

Dried saliva spot (DSS) is becoming increasingly popular in biomedical research, especially in the field of disease diagnosis. DSS testing has several advantages over traditional saliva testing methods. One major advantage is the stability of the dried sample. Unlike traditional liquid saliva samples that require immediate refrigeration or freezing to maintain their integrity, dried saliva samples are stable at room temperature for long periods, making them easier and more cost-effective to store and transport.

TASA Science has developed a novel, cost-effective, and simple method for rapidly and efficiently isolating RNA directly from dried saliva samples applied on any chemically treated filter paper, including Indicating FTA™ cards, Ahlstrom-Munksjö GenSaver™ Color 2.0 cards, etc.

The objective of this study is to evaluate the efficacy of releasing viral RNA from GenSaver Color 2.0 cards with TASA FastEx RNA Dried Saliva Spot Kit and investigate the stability of SARS-CoV-2 on GenSaver Color 2.0 cards for long-term RNA preservation at ambient temperatures.

MATERIALS AND METHODS

All saliva samples were collected from healthy individuals and spiked with SARS-CoV-2 culture fluid (Heat inactivated, Zetometrix™), fully synthetic SARS-CoV-2 RNA (Twist Bioscience™), QuickExtract™ DNA Extraction Solution (Lucigen) and GenSaver Color 2.0 Cards (Ahlstrom Munksjö).

dried_saliva_spot_workflow.png

Sample Collection and Storage

  1. Apply saliva sample spiked with SARS-CoV-2 culture fluid onto the GenSaver Color 2.0 card.

  2. Allow sample to dry completely (approximately 4 hours) and store the card in the multi-barrier pouches at ambient temperature in the dry-keeper desiccator cabinet.

TASA RNA Purification and Elution Protocol

  1. Punch 4-6 disks (6 mm diameter) or cut 1-2 strips (2.0 cm x 0.6 cm) from the Sample Collection Card and place them into a 1.5 mL microtube.  

  2. Add 1.0 mL of TASA Dried Saliva Spot Purification Solution 1 and vortex gently several times for approximately 3 minutes. Pipette the TASA Dried Saliva Spot Purification Solution 1 up and down to further mix, then remove as much solution as possible. 

  3. To wash, add 1.0 mL of Nuclease-free Water and vortex gently several times for approximately 2 minutes. Discard the water completely. Repeat this washing step one more time.

  4. Add 0.4 mL of TASA Dried Saliva Spot Purification Solution 2 and vortex gently several times for approximately 2 minutes. Pipette the TASA Dried Saliva Spot Purification Solution 2 up and down to further mix using the 1.0 mL pre-cut pipette tips or wide bore pipette tips, then remove the solution completely.

  5. Add 60-80 µL of TASA Rapid RNA Elution Solution 1.

  6. Set the heat block to 85°C. Incubate for 10 minutes to release the RNA. Centrifuge briefly.

  7. Add 6-8 µL of TASA Rapid RNA Elution Solution 2.

  8. To extract the RNA, press the punches or strips to the bottom of tube with 200 µL pre-cut pipette tips or wide bore pipette tips and recover the RNA solution.

      RNA is ready for use.

Note: Repeat steps 5-8 to obtain more RNA if necessary.

Real-time PCR Analysis

SARS-CoV-2 RNA yield is measured by amplifying the SARS-CoV-2 nucleocapsid gene N1 region and nucleocapsid gene N2 region, together with human RNase P nucleic acid as an endogenous internal control.

 

Primers and probe for N1:

  • N1-F1: GAC CCC AAA ATC AGC GAA AT

  • N1-R1: TCT GGT TAC TGC CAG TTG AAT CTG

  • N1-probe: FAM-ACC CCG CAT TAC GTT TGG TGG ACC -BHQ1.

 

Primers and probe for N2:

  • N2-F1: TTA CAA ACA TTG GCC GCA AA

  • N2-R1: GCG CGA CAT TCC GAA GAA 

  • N2-probe: FAM-ACA ATT TGC CCC: CAG CGC TTCAG-BHQ1.

 

Real Time PCR assay was performed with the Promega GoTaq® Probe 1- Step RT-qPCR System and the 7500 Fast Real-Time PCR System (Applied Biosystems). PCR conditions comprised of reverse transcription step at 45 °C for 5 minutes followed by an initial denaturation step at 95°C for 30 seconds and 45 cycles of denaturation at 95°C for 3 seconds, and annealing/elongation at 62°C for 30 seconds. 

 

When performing quantitative real-time PCR analysis, mean CT value was calculated using the average of 3 x PCR replicates. The Ct (threshold cycle) of the test sample was compared against a standard curve constructed using dilutions of the Twist synthetic SARS-CoV-2 RNA (1,000,000 copies/µL).

qRT-PCR Assay for Assessment of RNA Quality

TASA has developed a qRT-PCR assay using different amplicon lengths (small, medium, and large) to assess RNA integrity based on qPCR assays that produce 3 amplicons of different lengths from the same target.  The cDNA is amplified from the isolated RNA, producing amplicons of different lengths using one common primer.  The detection of all 3 sized fragments by qRT-PCR indicates high integrity RNA.

 

We established a qRT-PCR assay for SARS-CoV-2 that employs amplicon length to assess RNA integrity producing 3 amplicons of different lengths (small, medium and large) from the same target using one common primer.  The following primer pairs were used for the assay:   

Synthetic SARS-CoV-2RNA Extraction Efficiency

To determine RNA extraction efficiency, 5 µL of diluted solution of Twist synthetic SARS-CoV-2 RNA (10,000 copies/µL) were spotted directly onto a GenSaver Color 2.0 card and dried at RT for at least 4 hours. Using a 6 mm hole puncher, we performed 1, 2, 3, 4, 5, 6, and 7 punches, and placed each set into seven 2.0 mL microtubes (this was done in triplicate for a total of 21 different samples). Subsequently, the samples were processed using TASA FastEx RNA Dried Saliva Spot Kit. 

Concurrently, the same amount of Twist synthetic SARS-CoV-2 RNA was used directly for reverse transcription and qPCR. The efficiency of Synthetic SARS-CoV-2 RNA extraction from the punches was assessed by comparing the result derived from Twist synthetic SARS-CoV-2 RNA with reverse transcription and qPCR. 

SARS-CoV-2’s RNA recovery and stability on GenSaver Color 2.0 Card stored at ambient temperatures for an extended period

Saliva was spiked with various concentrations of heat-inactivated SARS-CoV-2 culture fluid (neat culture fluid, culture fluid/saliva 1:9, culture fluid/saliva 1:3) and was applied on GenSaver Color 2.0 Cards and stored in multi-barrier pouches at ambient temperature in the dry-keeper desiccator cabinet.  5 µL of neat culture fluid, 10 µL of diluted culture fluid 1:9, and 10 µL of culture fluid 1:3 was spotted onto GenSaver Color 2.0 cards. 

After storing the cards for a period of 1, 30, 60, 210, 240, and 300 days, we used one or two punches from the spotted GenSaver Color 2.0 cards for each experiment to investigate the recovery and stability of SARS-CoV-2 RNA on the GenSaver Color 2.0 cards. The samples were processed using TASA FastEx RNA Dried Saliva Spot Kit according to the TASA RNA Purification and Elution Protocol and analyzed by qRT-PCR.

As a reference method, 5 µL of neat culture fluid or 10 µL of diluted culture fluid were treated with 15 µL of QuickExtract™ DNA Extraction Solution (Lucigen) to extract RNA from SARS-CoV-2 culture fluid samples. The mixtures were heated for 10 min at 65°C and then for 2 min at 95°C. The extracted RNA from culture fluid samples were used for RT-PCR analysis.  

RESULTS

Synthetic SARS-CoV-2 RNA Extraction Efficiency

The results detailed below show the efficiency of Synthetic SARS-CoV-2 RNA extraction from the punches by comparing with the results derived from Twist synthetic RNA used directly for reverse transcription and qPCR.  

The data shown in Figure 1 illustrates the standard curve prepared with 10-fold serial dilutions of Twist synthetic SARS-CoV-2 RNA.

Figure 1:

The data shown in Figure 2 illustrates that the RNA recovery from GenSaver 2.0 Color cards increases with increasing number of punches.  TASA FastEx RNA Dried Saliva Spot Kit enables consistent recovery of RNA, as shown on the graph.

Figure 2:

The data shown in Figure 3 illustrates that the RNA recovery from GenSaver cards increases with increasing number of punches. Diluted Reference Method data points were calculated using Twist Synthetic RNA.

Figure 3:

SARS-CoV-2 RNA Recovery and Stability on GenSaver Color 2.0 Cards Stored at Ambient Temperature for up to 300 Days

The data in Table 1 referenced below illustrates the recovery and stability on GenSaver Color 2.0 cards obtained from Real Time PCR analysis during a period of 300 days. 

Table 1:

P1 Table 1.png

SARS-CoV-2 RNA Recovery and Stability on GenSaver Color 2.0 Cards Stored at Ambient Temperature for up to 300 Days

A. SARS-CoV-2 RNA Recovery from GenSaver card  

The efficiency of SARS-CoV-2 RNA recovery from GenSaver cards with TASA FastEx RNA Dried Saliva Spot Kit was assessed by comparing results derived from neat culture fluid and diluted culture fluid extracted RNA samples with reverse transcription-and qPCR.

 

Table 1 shows the Ct values of the released RNA from GenSaver card compared to the Ct values of the extracted RNA original culture fluid samples.  ΔCt values varied from 1.52 to 3.28 indicating efficient RNA recovery from GenSaver cards with TASA FastEx RNA Dried Saliva Spot Kit.

 

B. Sars-CoV-2 RNA Stability on GenSaver card 

Table 1 shows the mean Ct values were obtained from Real Time PCR analysis during a period of 300 days.  SARS-CoV-2 RNA was still being detected using real-time PCR analysis even after 300 days.  

 

Figure 4 illustrates that there were no significant changes in cycle threshold values over the 300-day period at ambient temperature, suggesting stability of viral SARS-CoV-2 RNA on GenSaver Color 2.0 cards and indicating consistent recovery of the viral RNA using TASA FastEx RNA Dried Saliva Spot Kit.

Figure 4:

F4 P1 SARS-CoV-2 RNA Stability.png

C. Assessment of Isolated RNA Quality

Successful reverse-transcription polymerase chain reaction (RT-PCR) was demonstrated in all samples with RNA extracted from GenSaver cards using the TASA FastEx RNA Dried Saliva Spot Kit. Detection of all 3 sized fragments (71 bp, 243 bp, and 831 bp) using qRT-PCR indicated RNA integrity and high-quality RNA retrieved from the GenSaver cards. The PCR products were submitted for Sanger sequencing and the 2 amplicons of 243 bp and 831 bp were confirmed.    

CONCLUSION

This study shows that the TASA FastEx RNA Dried Saliva Spot Kit simplifies and accelerates RNA purification, offering greater efficiency and shortening the processing time to just 30 minutes from start to finish. Our kits can yield sufficient high-quality RNA from GenSaver Color 2.0 cards for all molecular downstream applications, including Real-time qRT-PCR.

 

This study also shows that viral SARS-CoV-2 RNA can be recovered efficiently from GenSaver Color 2.0 cards stored at ambient temperatures for up to 10 months, enabling long-term RNA preservation.  GenSaver Color 2.0 cards can be used for storing and transporting the samples at ambient temperatures for long durations of time.

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