Figure 3. Relative detection sensitivity of MALAT1 -- a potential lncRNA biomarker for several different types of cancer
include cyclin dependent kinase 1 (CDC2), Insulin Like Growth Factor Binding Protein 5 (IGFBP5) and Midkine (MDK). Bladder Cancer Markers In the following study, Meridian’s Air- Dryable ™ Direct RNA/DNA qPCR Urine (MDX151) was compared to UltraPlex ™ 1-Step ToughMix for its ability to amplify bladder cancer makers CDC2, IGFBP5 and MDK in samples containing 10% urine (Figure 4). Specifically, reactions were set up with 10% DNase-treated-human urine and 200 pg of CDC2, IGFBP5 or MDK RNA and run either with air-dried Air-Dryable ™ Direct RNA/DNA qPCR Urine or wet UltraPlex ™ 1-Step ToughMix on a QuantStudio using 50°C for 10 minutes, 95°C for 2 minutes and 50 cycles of 95°C for 10 seconds and 60°C for 20 seconds. Across all three markers, Air-Dryable ™ Direct RNA/DNA qPCR Urine showed a lower ct value demonstrating that it is able to detect cancer related RNA markers from urine faster and with higher sensitivity compared to other mixes. Prostate Cancer Markers Currently, the gold-standard screening assay for prostate cancer is the PSA (prostate specific antigen) test, and elevated levels that suggest the presence of cancer are further examined by transrectal or trans- perineal biopsy. Although this strategy has proven to be a very effective diagnostic algorithm, cancer detection rates from tissue biopsy are low, varying between 36% - 54% as not all elevated PSA antigen results are caused by cancer 11 . In addition, repeat biopsies are often required and there is a risk of severe infection and hospitalization with each procedure.
QuantStudio using 50°C for 10 minutes, 95°C for 3 minutes and 40 cycles of 95°C for 10 seconds and 60 °C for 25 seconds (Fig. 3) . Direct detection of molecular biomarkers from urine Advances in liquid biopsy have largely centred around blood specimens, however other body fluids such as stool, saliva and urine can also be used. In particular, urine has demonstrated great promise in urological cancers and non-urological cancers such as prostate cancer 9 and offers significant benefits over blood as it is completely non-invasive, can be collected in large volumes, and is a rich source for ctDNA. Several liquid biopsy assays based on qPCR methods for bladder cancer detection and monitoring are already FDA-approved and have helped to lower the economic burden of bladder cancer patient surveillance and to increase patient compliance 10 . Most of the available tests examine a panel of mRNA biomarkers whose overexpression has been linked stages 1-4 of bladder cancer, and Figure 3: Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is one of the few well-studied long non-coding RNAs (lncRNAs), which presents high expression and enhanced activity in numerous tumor tissues and cells, playing a critical role in tumor invasion and metastasis. In this study, the sensitivity of Air-Dryable ™ Direct RNA/DNA qPCR Blood (MDX121) (red) to detect MALAT1 was compared against QuantaBio UltraPlex ™ 1-Step ToughMix in the presence of 5% DNase-treated-serum. The results illustrate the greater sensitivity of MDX121 with serum, suggesting its greater suitability lncRNA biomarker screening.
Direct detection of lncRNA biomarkers from blood - Detecting early-stage cancers using lncRNA biomarkers LncRNAs are defined as RNA transcripts greater than 200 nucleotides in length that have no or limited protein-coding potential. Research has shown that lncRNA, exert distinct biological functions through diverse molecular mechanisms 6 . Several studies have validated the use of exosomal lncRNAs as minimally invasive diagnostic and prognostic markers in several types of cancers. MALTA1(metastasis associated in lung adenocarcinoma transcript 1) is a 8.8kb long non-coding RNA (lncRNA) that has attracted significant attention in the past couple of years as a potential biomarker for cancer diagnosis as well as prognosis. MALAT1 is widely expressed in normal tissues and is aberrant expressed in cancers, suggesting that it plays a role in cancer pathophysiology and disease progression 7 . Studies examining diagnostic testing for lung-cancer associated malignant pleural effusion (LC-MPE), have found that combinations of MALAT1 and carcinoembryonic antigen (CEA), a tumor marker used in the detection of several cancers including lung cancer, provided higher sensitivity and accuracy in predicting LC-MPE than CEA alone 8 . In the following study, Meridian’s Air-Dryable Direct DNA qPCR Blood was tested in the presence of 5% DNase-treated-serum (to remove genomic DNA for RNA detection) for its sensitivity to detect MALAT1. DNase I was added to serum and incubated for 2 h at 37°C followed by heat inactivation at 95°C for 10 minutes, and reactions set up with 5% of this DNase-treated-serum and known copy number concentrations of the long non- coding RNA target (MALAT1), and run with either Air-Dryable Direct DNA qPCR Blood or UltraPlex ™ 1-Step ToughMix on a
Figure 4. Detection of tumor-related mRNA markers (CDC2 kinase, IGFBP5 and MDK) in samples containing 10% urine
A) CDC2
B) IGFBP5
C) MDK
Figure 4: Meridian’s Air-Dryable ™ Direct RNA/DNA qPCR Urine (red) was compared to UltraPlex ™ 1-Step ToughMix (grey)for its ability to amplify bladder cancer makers CDC2, IGFBP5 and MDK in samples containing 10% urine. Specifically, reactions were set up with 10% DNase-treated-human urine and 200 pg of CDC2, IGFBP5 or MDK RNA and run either with air-dried Air-Dryable ™ Direct RNA/ DNA qPCR Urine or wet UltraPlex ™ 1-Step ToughMix on a QuantStudio using 50°C for 10 minutes, 95°C for 2 minutes and 50 cycles of 95°C for 10 seconds and 60°C for 20 seconds. Across all three markers, Air-Dryable ™ Direct RNA/DNA qPCR Urine showed a lower ct value demonstrating that it is able to detect cancer related RNA markers from urine faster and with higher sensitivity compared to other mixes.
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