Stability of RNA Preserved in Norgen's Saliva RNA Preservative at room temperature for up to 2 months. Saliva samples were collected from numerous donors and mixed, and then an equal volume of Norgen's Saliva RNA Preservative was added to the saliva. The preserved saliva was then stored at room temperature for up to 2 months. Saliva RNA was subsequently isolated at day 0, day 4, day 6, day 16, 1 month and 2 months from 0.25 mL of the saliva/preservative sample using the supplementary protocol associated with Norgen's Total RNA Purification Kit (Cat. 17200). For visual analysis, 7.5 µL from 50 µL elution was mixed with RNA loading dye and was run on a 1x MOPS agarose gel. RNA integrity was maintained after the saliva samples were stored for up to 2 months at ambient temperatures in Norgen’s Saliva RNA Preservative.

Stability of three house keeping genes over a 2 month period. GAPDH, Human 18S rRNA and beta actin genes were monitored from saliva samples stored at ambient temperature in Norgen's Saliva RNA Preservative. RNA was isolated from 250 µL of preserved samples on day 0, day 4, day 6, day 10, day 16, day 30 and day 60 using Norgen's Total RNA Purification Kit (Cat. 17200). Next, 4 µL of elution was used in 20 µL of the real-time PCR reaction (SYBR Green I) using CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad). All of the genes were preserved and stable over 2 month period.

Norgen’s Saliva RNA Preservative is able to successfully preserve saliva RNA samples when used outside of the ideal range of 1:1 at room temperature for 3 days. As the amount of saliva added to 2 mL of Saliva RNA Preservative increased from 0.2 mL to 3 mL, RNase protein and β-actin gene were monitored for RNA stability using one-step RT-PCR. The results indicated that the variation of the saliva input volumes did not significantly influence the RNA gene detection.

Detection of Viral, Bacterial and Human RNA from Preserved Saliva Samples. Saliva (2 mL) collected from a single healthy donor was mixed with 2 mL of Saliva RNA Preservative. Norgen’s COVID-19 positive control from the 2019-nCoV TaqMan RT-PCR Kit (E/RdRP gene) (Cat. TM67200) was spiked into the preserved saliva along with HeLa cells (1.6 m cells /ml ) to generate a contrived sample. From the contrived sample, total RNA was purified using Norgen’s Total RNA Purification Kit (Cat. 17200) and 5 genes (16s rRNA, COVID-19 E gene, RdRP-P1 and P2 gene and RP) were analyzed by PCR or RT-PCR to detect viral, bacterial and human genes.

Stability of RNA and DNA stored in Norgen's Total Nucleic Acid Preservative. GAPDH (for DNA) and β-actin (for RNA) were monitored over an 18 week period for DNA and 60 days for RNA from swab samples stored at ambient temperature in Norgen’s Total Nucleic Acid Preservation Tubes. RNA was isolated from 250 µL of preserved swab samples on day 0, day 4, day 6, day 10, day 16, day 30 and day 60 using Norgen’s Total RNA Isolation Kit (Cat.# 17200). DNA was isolated from 250 µL of preserved swab samples on day 0 and 18 weeks using Norgen’s Saliva DNA Isolation Kit (Cat# Dx45400). Next, 4 µL of elution was used in a 20 µL real-time PCR reaction using CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad). RNA can be stored in Norgen’s Total Nucleic Acid Preservative for up to 60 days and DNA can be stored for up to 18 weeks at room temperature with no change in quality and quantity of RNA and DNA.

Stability of RNA from different swab sample types stored in Norgen's Total Nucleic Acid Preservative. Amplification of the RNase Protein (RP) gene from 5 oropharyngeal swab samples and 5 nasopharyngeal swab samples purified using Norgen’s Total RNA Purification Kit were successfully validated from both swab sample types with high efficiency (<40 Ct.) by TaqMan One-Step RT-PCR.

Stability of DNA from different swab sample types stored in Norgen's Total Nucleic Acid Preservative. Amplification of the GAPDH gene from 5 oropharyngeal swab samples and from 5 nasopharyngeal swab samples purified using Norgen's Saliva DNA Isolation Kit were successfully validated from both swab sample types with high efficiency (<40 Ct.) by TaqMan PCR.

Inhibitory Effects of Norgen’s Total Nucleic Acid Preservative on the Growth of Bacteria, Yeast and Viruses. The effect of Norgen’s Total Nucleic Acid Preservative on the growth of microorganisms clearly demonstrated that Norgen’s preservative inhibits the growth of virus, bacteria and yeast, thereby resulting in non-infectious samples that can be handled and shipped safely.

Downstream Application of Preserved Swab RNA & DNA. The application of the RNA or DNA purified from preserved oropharyngeal swab samples was evaluated to detect RNase P (house keeping gene) and 16S rRNA (bacterial DNA) from swab samples collected from 60 donors and the preserved at room temperature overnight. Each individual swab sample contains different levels of cells, contributing to the variable Ct of each gene detection, but all passed the acceptance criteria for valid RT-PCR and PCR (< 35Ct, red line) in the 20 µL reaction, indicating that preserved swab RNA and DNA stored in Norgen’s Total Nucleic Acid Preservative is of high quality for downstream applications, such as real-time RT-PCR and PCR.

High Quality of Isolated RNA with Complete Size Range. Unlike most competitors' kits, Norgen's Total RNA Purification Kit Dx allows for the isolation of all sizes of RNA, from the very large RNA down to the microRNA, without the use of phenol. Total RNA was isolated from 1 x 109 E. coli cells using Norgen’s Total RNA Purification Kit and a competitor’s kit. Five microliters and 1 µL of the 50 µL isolated RNA was analyzed on an agarose gel (Panel A) and the Agilent® 2100 BioAnalyzer RNA Nano 6000 chip (Panel B), respectively. Note the presence of small RNA species (red square) in the samples isolated via Norgen's kit and the absence of these RNA species in the competitor RNA preparation.

Amplification of Both Large and Small RNAs in the Same Extraction. Norgen's Total RNA Purification Kit Dx isolates the complete size range of RNA, including small RNAs without the use of phenol. Total RNA was isolated from 0.75 million HeLa cells using Norgen's Total RNA Purification Kit Dx, various silica-based competitors and phenol-based TRI Reagent. RT-PCR was performed according to Shi and Chiang (2005). Fifteen microliters of the 50 µL isolated RNA were polyadenylated in a 50 µL Poly-(A)-Polymerase reaction. Four microliters of the polyadenylated RNA were used in a 20 µL reverse transcription reaction with a poly T adaptor primer. One microliter of the reverse transcription was used in a 20 µL qPCR reaction with primers against the human microRNAs (miR-19 and miR-21) and large mRNA (S15). Only the use of Norgen's Total RNA Purification Kit Dx resulted in detection of both microRNAs and mRNA amplification products similar to Competitor 2's kit but without the use of phenol.

High Quality of RNA from a Diverse Range of Inputs. Norgen's Total RNA Purification Kit Dx allows RNA isolation from a wide range of species or tissue types. Total RNA was isolated from 5 x 108 E. coli cells, 1 x 106 HeLa cells, 100 µL rat blood and 10 mg hamster kidney using Norgen's Total RNA Purification Kit Dx. One microliter of the 50 µL isolated RNA was analyzed on the Agilent® 2100 BioAnalyzer using an RNA Nano 6000 chip. Note the integrity of RNA from all inputs with the presence of small RNA species. Norgen's Total RNA Purification Kit Dx consistently isolates high quality RNA from various inputs that score a RIN value between 8 and 10.

Great Isolation Sensitivity. Norgen's Total RNA Purification Kit Dx allows sensitive RNA extraction from as little as a single cell. Total RNA was extracted from a decreasing number of 293 HEK cells from 1 million cells down to a single cell. Five microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using oligo dT primer. Three microliters of the reverse transcription was used in a 20 µL PCR reaction with primers to detect the human beta-actin transcripts. PCR products of beta-actin were detected from as little as a single cell. M is the marker lane.

Linear and Sensitive Isolation of Both Large and Small RNA. Norgen’s Total RNA Purification Kit Dx allows consistent isolation of both large and small RNA from different input amounts. Total RNA was isolated from 10 to 100,000 HeLa cells using Norgen's Total RNA Purification Kit Dx (blue), a competitor’s silica-based kit (green) and a phenol-based RNA extraction method (red). Relative expression of miR-21 (Panel A), and S15 (Panel B) was determined by RT-qPCR of total RNA samples. In brief, one microliter of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Two microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21 (Panel A) and the S15transcripts (Panel B). The resulting threshold cycle (Ct) values were plotted against input cell number. RNA isolated using Norgen's Total RNA Purification Dx had the best linearity (higher R2) and sensitivity (lower Ct) for both large RNA (S15) and small RNA (miR-21).

Effective and Consistent Detection of miRNA from Plasma. Norgen's Total RNA Purification Kit Dx can effectively isolate miRNA from plasma. Total RNA was isolated from 50, 100 or 200 µL of rat plasma in triplicates using Norgen's Total RNA Purification Kit Dx (blue), a competitor’s silica-based kit (green) and a phenol-based RNA extraction method (red). Stem loop RT-qPCR using primers specific to miR-21 was performed. In brief, two microliters of the 50 µL isolated RNA was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer or oligo dT primer. Three microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21. Norgen's Total RNA Purification Kit Dx is the only product that showed (1) consistent detection of miR-21 transcripts across all input volumes, and (2) Ct values correlated to input volume (decrease Ct with increase input).

Recovering Diverse miRNA Species from Plasma with Better Consistency. Norgen's Total RNA Purification Kit Dx isolates plasma RNA that performs more consistently in downstream applications such as microarrays. Total RNA including miRNA was isolated from 100 µL of mouse plasma in duplicate using Norgen's Total RNA Purification Kit Dx, Competitor 1's leading miRNA Kit or a phenol-based RNA extraction method. One hundred nanograms of extracted RNA from each kit was applied to an Illumina microRNA expression profiling kit. Scatter plots display better consistency (better clustering) of replicate signals from Norgen's samples. Genes with Pval < 0.01 for both replicates were in blue. The associated table suggested that Norgen's protocol recovered the same diversity of miRNA with better consistency (higher r2 value).

Better Diversity of miRNA Detected from Plasma. Norgen's Total RNA Purification Kit Dx isolates miRNA from plasma with better diversity than a leading competitor. Total RNA including miRNA was isolated from 100 µL of plasma using Norgen's Total RNA Purification Kit Dx or 625 µL of plasma using Competitor A's leading miRNA Kit, and was applied to an NCode expression profiling kit. Microarray images suggested that Norgen's Total RNA Purification Kit Dx (left) isolates a better diversity of miRNA from smaller input amount of plasma than the competitor’s miRNA kit (right). Image courtesy of LC Sciences, Houston.

Better Diversity of miRNA Detected from HeLa Cells using Illumina Small RNA Next Gen Sequencing. Norgen's Total RNA Purification Kit Dx isolates miRNA from HeLa cells with better diversity than a leading competitor. Total RNA including miRNA was isolated from 1 million HeLa cells using Norgen's Total RNA Purification Kit Dx and Competitor Q's leading miRNA Kit, and was applied to Illumina Small RNA Next Gen Sequencing on a MiSeq sequencer. Panel A showed that Norgen’s Total RNA Purification Kit Dx recovers a higher number of miRNAs than the competitor. In particular, the higher diversity is achieved with a faster and simpler procedure in as little as 20 minutes of RNA sample preparation time without the use of phenol. Panel B is a scatter plot of the average RPM (reads per million) of the miRNAs detected used to compare Norgen and the competitor’s kit recovery of miRNA. Norgen’s Total RNA Purification Kit Dx recovered a significantly higher number of miRNAs that have higher RPM, as summarized in the graph insert as well as in Panel C.

SARS-CoV-2 E-gene and RdRP Amplification from Preserved Swabs and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit. 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 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).

RNase P Gene Amplification of Fresh and Preserved Swab and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit. 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. Preserved Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices. 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 (Cat. #Dx69100), 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.

Human miR-21 Amplification of Fresh and Preserved Swab and Saliva RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit. 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. Preserved Saliva samples were collected into Norgen’s Saliva RNA Collection and Preservation Devices. 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 (Cat. #Dx69100), 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.

Average RNA Yield of Samples Isolated by Norgen’s Saliva/Swab RNA Purification Kit. The average yield of RNA isolated by Norgen’s Saliva/Swab RNA Purification Kit (Cat. #Dx69100) 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 (Lot #598129) and preserved saliva samples were collected using Norgen’s Saliva RNA Collection and Preservation Devices. 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.

Duplex TaqMan RT-PCR analytical sensitivity for each of the SARS-CoV-2 targets based on the Charité/Berlin protocol. The analytical sensitivity was determined by analyzing six serial dilutions (200,000 copies to 2 copies) of Norgen's positive control from the COVID-19 TaqMan RT-PCR Kit (E/RdRP gene)(Cat. TM67200). Five µL of template was used in a 20 µL of the one-step RT-PCR reaction and the arrow indicates a copy number of 10.

To test the analytical specificity of Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP genes), the E/RdRP/RP Positive Control was used to test the kits specificity against RNA purified from other related pathogens. RdRP reactions were only prepared for samples required for confirmation and discriminatory detection based on the first gene detection result. Amplification was measured by real time RT-PCR. As can be seen, only Norgen’s E/RdRP/RP Positive Control showed amplification for all genes. COVID-19 WA showed amplification of the E gene and the two RdRP gene targets. SARS-CoV showed amplification only with E gene and RdRP Discriminatory. None of the remaining pathogens showed amplification with any of the tested genes.

Clinical testing of the COVID-19 TaqMan RT-PCR Kit (E/RdRP gene)(Cat. TM67200) was conducted with contrived oropharyngeal swabs (circle) and nasopharyngeal swabs (cross). Swab samples were collected from healthy donors and placed into preservative using Norgen’s Total Nucleic Acid Preservation Tubes (Cat. 69200). To prepare the contrived positive samples, the preserved swab samples were spiked with 100,000 copies of positive control. Total viral RNA was then isolated using Norgen’s Total RNA Purification Kit (Cat. 17200). Following RNA isolation, one-step RT-PCR for detection of the four targets was performed using Norgen’s COVID-19 TaqMan RT-PCR Kit (E/RdRP gene) (TM67200).

Example of SARS-CoV-2 detection using Norgen's 2019-nCoV TaqMan RT-PCR Kit Dx. One-Step RT PCR was prepared according to the manual provided with the kit. Five uL of Norgen's positive control (2x 10^6 theoretical copy number) was used for the 25 uL PCR reaction. Each target indicated by the name was successfully detected including the internal control (RP).

Analytical sensitivity. The limit of detection of the 2019-nCoV TaqMan RT-PCR Kit Dx was determined using 20 contrived samples of each sample type. Contrived nasopharyngeal swabs (A), oropharyngeal swabs (B) and saliva samples (C) were generated by spiking 2019-nCoV RT-PCR Positive Control corresponding to 10 copies per PCR reaction. Confirmatory results were acceptable at a 95% confidence interval. This can be achieved when obtaining a minimum of 19 positive samples out of the 20 samples spiked at the limit of detection. As seen in Table 1, the limit of detection of Norgens 2019-nCoV TaqMan RT-PCR Kit Dx from RNA isolated from nasopharyngeal swabs, oropharyngeal swabs and saliva samples was confirmed to be 10 copies per PCR reaction at a 95% confidence interval.

Specificity analysis. To test the analytical specificity of Norgens 2019-nCoV TaqMan RT-PCR Kit Dx, the 2019-nCoV RT-PCR Positive Control Dx (containing the two nCoV nucleocapsid target gene RNA (N1 and N2) and RNase P) was used to test the kits specificity against RNA purified from other related pathogens. As can be seen in the table only Norgens 2019-nCoV RT-PCR Positive Control showed amplification for all genes. Therefore, Norgens 2019-nCoV TaqMan RT-PCR Kit Dx can be used to specifically detect, confirm and discriminate COVID-19.

Diagnostic Accuracy. Clinical evaluation of the accuracy of the 2019-nCoV TaqMan RT-PCR Kit Dx was conducted with contrived nasopharyngeal swabs, oropharyngeal swabs and saliva samples by testing 30 positive and 30 negative samples to generate the Positive Percentage Agreement (PPA), Negative Percentage Agreement (NPA) and overall percentage agreement (OPA) as a measurement of estimated Diagnostic Accuracy. For the 30 contrived positive samples, each were spiked with 5 µL of different concentrations of the 2019-nCoV RT-PCR Positive Control to generate input 15 samples of variable transcript content that corresponds to a limit of detection (LoD) range from 1X to 1,000X (10 samples at 1X, 10 samples at 2X, 4 samples at 10X, 3 samples at 100X and 3 samples at 1,000X). The remaining 30 samples from each sample type were not spiked (nonreactive). RNA isolation was performed from all samples using Norgen’s Saliva/Swab RNA Purification Kit (Cat. #69100) and RNA was eluted in 50 µL. Five microliters of the isolated RNA were used as a template in the Norgen’s 2019-nCoV TaqMan RT-PCR Kit Dx to detect the 3 targets of the kits (N1, N2 and RP). The various SARS-CoV-2 kit targets can be detected from RNA isolated from contrived nasopharyngeal swabs, oropharyngeal swabs and saliva samples, at various detection limits using Norgen’s 2019-nCoV TaqMan RT-PCR Kit Dx with no detectable viral targets from non-reactive samples.

Interpretation of Assay Results. The Negative Control (NTC No Template Control) reaction(s) must be negative and not exhibit fluorescence growth curves that cross the threshold line. If there is any amplification with the NTC the run is not valid and no interpretation of 2019-nCoV detection can be made. In this case the assay must be repeated. The Positive Control (PosC) reaction(s) should produce a positive result with an expected Ct value below 20 for each target. If the positive control does not provide a positive result the run is not valid and no interpretation of 2019-nCoV detection can be made and the assay must be repeated. If the NTC and PosC are exhibiting the correct results, the results of the detection assays can be interpreted as outlined in the product manual.