Saliva/Swab RNA Purification Kits

Supporting Data

Figure 1. SARS-CoV-2 E-gene and RdRP Amplification from Preserved Swabs and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100). Duplicate Nasopharyngeal swabs, Oropharyngeal Swabs and Saliva samples were collected from different donors. Nasopharyngeal and Oropharyngeal swab samples were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat. 69200). Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices (Cat. RU53800). All collected samples were spiked with 3 different concentrations from Norgen’s E/RdRP/RP Positive Control (included in Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes – TM67200) and processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100), using an input of 0.25 mL from each preserved samples. The sensitivity of the isolation method was assessed by amplifying the spiked SARS-CoV-2 spiked targets using Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes (TM67200). Figure 1 shows the linear recovery of the different spike-in amounts indicating the high sensitivity of the isolation procedure as well as the high quality of the RNA purified using Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100).

Figure 2. RNase P Gene Amplification of Fresh and Preserved Swab and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100). Duplicate preserved (P) and non-preserved (NP) Nasopharyngeal swabs, Oropharyngeal Swabs, and Saliva samples were collected from different donors. Preserved Nasopharyngeal and Oropharyngeal swab samples were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat #69200). Preserved Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices (Cat #RU53800). Non-preserved samples were processed immediately without mixing with any preservative. All collected samples were processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification Kit, using an input of 0.25 mL from preserved sample or the entire non-preserved swab for the non-preserved conditions. Non-preserved saliva samples were isolated from a 0.25mL input as well. Isolated RNA was tested by amplification of the RNase P gene by RT-PCR.

Figure 3. Human miR-21 Amplification of Fresh and Preserved Swab and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100). Duplicate preserved (P) and non-preserved (NP) Nasopharyngeal swabs, Oropharyngeal Swabs, and Saliva samples were collected from different donors. Preserved Nasopharyngeal and Oropharyngeal swab samples were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat #69200). Preserved Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices (Cat #RU53800). Non-preserved samples were processed immediately without mixing with any preservative. All collected samples were processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification Kit, using an input of 0.25 mL from preserved samples or the entire non-preserved swab for the non-preserved conditions. Non-preserved saliva samples were isolated from a 0.25mL input as well. Isolated RNA was tested by amplification of the human miR-21 detected by stem-loop RT-PCR.

Figure 4. Average RNA Yield of Samples Isolated by Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100). The average yield of RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit is ≥ 1 µg. Duplicate preserved (P) and non-preserved (NP) swab (nasal and oral) and saliva samples were collected from different donors. Preserved swab samples (nasal or oral) were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat #69200) and preserved saliva samples were collected using Norgen’s Saliva RNA Collection and Preservation Devices (Cat #RU53800). Non-preserved samples were processed immediately without mixing with any preservative. All collected samples were processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification Kit, using an input of 0.25 mL from preserved samples and the non-preserved saliva or the entire non-preserved swab for the non-preserved swabs. NanoDrop was used to estimate the concentration and yield of the isolated RNA.

Figure 5. SARS-CoV-2 E-gene and RdRP Amplification from Preserved Swabs and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification 96-Well Kit (Cat. #69300). Duplicate Nasopharyngeal swabs, Oropharyngeal Swabs, and Saliva samples were collected from different donors. Nasopharyngeal and Oropharyngeal swab samples were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat. 69200). Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices (Cat. RU53800). All collected samples were spiked with 3 different concentrations from Norgen’s E/RdRP/RP Positive Control (included in Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes – TM67200) and processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification 96-Well Kit (Cat. #69300), using an input of 0.25 mL from each preserved sample. The sensitivity of the isolation method was assessed by amplifying the spiked SARS-CoV-2 spiked targets using Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes (TM67200). Figure 1 shows the linear recovery of the different spike-in amounts indicating the high sensitivity of the isolation procedure as well as the high quality of the RNA purified using Norgen’s Saliva/Swab RNA Purification 96-Well Kit (Cat. #69300).

Figure 6. RNase P Gene Amplification of Fresh and Preserved Swab and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification 96-Well Kit (Cat. #69300). Duplicate preserved (P) and non-preserved (NP) Nasopharyngeal swabs, Oropharyngeal Swabs, and Saliva samples were collected from different donors. Preserved Nasopharyngeal and Oropharyngeal swab samples were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat #69200). Preserved Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices (Cat #RU53800). Non-preserved samples were processed immediately without mixing with any preservative. All collected samples were processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification 96-Well Kit, using an input of 0.25 mL from preserved samples or the entire non-preserved swab for the non-preserved conditions. Non-preserved saliva samples were isolated from a 0.25mL input as well. Isolated RNA was tested by amplification of the RNase P gene by RT-PCR.

Figure 7. Human miR-21 Amplification of Fresh and Preserved Swab and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification 96-Well Kit (Cat. #69300). Duplicate preserved (P) and non-preserved (NP) Nasopharyngeal swabs, Oropharyngeal Swabs, and Saliva samples were collected from different donors. Preserved Nasopharyngeal and Oropharyngeal swab samples were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat #69200). Preserved Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices (Cat #RU53800). Non-preserved samples were processed immediately without mixing with any preservative. All collected samples were processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification 96-Well Kit, using an input of 0.25 mL from preserved samples or the entire non-preserved swab for the non-preserved conditions. Non-preserved saliva samples were isolated from a 0.25mL input as well. Isolated RNA was tested by amplification of the human miR-21 detected by stem-loop RT-PCR.

Figure 8. Average RNA Yield of Samples Isolated by Norgen’s Saliva/Swab RNA Purification 96-Well Kit (Cat. #69300). Duplicate preserved (P) and non-preserved (NP) swab (nasal and oral) and saliva samples were collected from different donors. Preserved swab samples (nasal or oral) were collected into Norgen’s Total Nucleic Acid Preservative Tubes (Cat #69200) and preserved saliva samples were collected using Norgen’s Saliva RNA Collection and Preservation Devices (Cat #RU53800). Non-preserved samples were processed immediately without mixing with any preservative. All collected samples were processed for RNA isolation using Norgen’s Saliva/Swab RNA Purification 96-Well Kit, using an input of 0.25 mL from preserved samples and the non-preserved saliva or the entire non-preserved swab for the non-preserved swabs. NanoDrop was used to estimate the concentration and yield of the isolated RNA.

Kit Specifications
Sample Volume Range	250 μL
Size of RNA Purified	All sizes, including small RNA (<200 nt)
Minimum Elution Volume	50 μL
Maximum Elution Volume	100 μL
Time to Complete 10 Purifications	15 - 20 minutes
Average Yield	≥ 1 μg * *Varies from sample to sample

Storage Conditions and Product Stability

All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Kit Component	Cat. 69100 (50 preps)	Cat. 69300 (192 preps)
Lysis Buffer A	30 mL	100 mL
Solution WN	18 mL	55 mL
Wash Solution A	18 mL	2 x 38 mL
Elution Solution A	6 mL	20 mL
Mini Spin Columns	50	-
Collection Tubes	50	-
Elution Tubes (1.7 mL)	50	-
96-Well Isolation Plate	-	2
96-Well Collection Plate	-	2
96-Well Elution Plate	-	2
Adhesive Tape	-	4
Product Insert	1	1

Automation

Saliva/Swab Automated RNA Purification Tutorial

Supplementary Protocol

Supplementary Protocol - Automated Procedure for Saliva/Swab RNA Purification and Concentration

Application Note

Application Note - Comparing the Isolation Efficiency of Norgen’s Saliva/Swab RNA Purification Kit on Hamilton Vantage and QIAsymphony DSP Virus/Pathogen Kit

Using Norgen’s Saliva/Swab RNA Purification Kit on the Hamilton Vantage to Isolate RNA from Fresh and Preserved Saliva Samples without Contamination

FAQs

Spin Column

Why do I have poor RNA recovery?

Poor RNA recovery could be due to one or more of the following:

Incorrect lysate preparation method:
Separate lysate preparation instructions need to be followed depending on the sample (Swab sample / fresh OR preserved saliva).
Column has become clogged.
Do not exceed the recommended input amount of saliva or swab sample. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels.
An alternative Elution Buffer was used.
It is recommended that the Elution Solution A supplied with this kit be used for maximum RNA recovery.
Ethanol was not added to the lysate.
Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.
Ethanol was not added to the Solution WN and Wash Solution A.
Ensure that 90 mL of 96-100% ethanol is added to the supplied Wash Solution A prior to use.

Why is the RNA degraded?

RNA can be degraded due to following factors:

RNase contamination.
RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to “Working with RNA” at the beginning of this user guide.
Improper storage of the purified RNA.
For short term storage RNA samples may be stored at –20°C for a few days. It is recommended that samples be stored at –70°C for longer term storage.
DNase used may not be RNAse-free.
Ensure that the DNase being used for the optional On Column DNA Removal step is RNase-free, in order to prevent possible problems with RNA degradation.

Why is the RNA not performing well in downstream applications?

If the RNA does not perform well in downstream applications, It may be due to one or more of the following:

RNA was not washed with Solution WN and Wash solution A.
Traces of salt from the binding step may remain in the sample if the column is not washed with Solution WN and 2 times with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the column.
Ethanol carryover.
Ensure that the dry spin under the Column Wash procedure is performed, in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

Saliva/Swab RNA Purification Kits