One of the most exciting potential applications of circulating non-coding RNAs like microRNA (miRNA) and long non-coding RNA (lncRNA) is as biomarkers for early detection of cancer. Cancer treatment, like treatment of many other diseases, can be far more effective when started early. Easily accessible markers like RNAs in the blood are an attractive possibility for finding cancers as soon as possible.
Numerous translational studies have focused on determining which miRNAs and lncRNAs are altered during cancer progression. Our scientists recently tested the effectiveness of qPCR for investigating these questions in non-small-cell lung cancer. Read on to see what they found!
Serum miRNA and lncRNA detection as a potential biomarker of lung cancer (a QIAGEN application note)
Authors: Song Tian, Jonathan M. Shaffer, Samuel Rulli, Brian Dugan, and Eric Lader
QIAGEN Sciences Inc., Frederick, MD, USA
Lung cancer is one of the leading causes of mortality globally, and the second most common cancer among both men and women in the United States. Although it has been extensively studied for years, there is still a great deal unknown that relates to its detection for clinical diagnosis, prognosis and mechanism.
The discovery of cancer biomarkers, specific molecules that help distinguish between normal and malignant conditions, may potentially be utilized to develop a more effective diagnostic tool for lung cancer. To explore the possibility of using serum miRNAs and lncRNAs as lung cancer biomarkers, we used the miScript PCR System and the human RT² lncRNA Cancer PathwayFinder PCR Array to evaluate cancer-related miRNA and lncRNA levels, respectively, in non-small cell lung cancer (NSCLC) patient serum samples.
Early diagnosis of lung cancer is still the key factor related to prognosis after treatment. Detection of lung cancer through traditional methods is limited in its effectiveness for early detection. Currently, the only recommended screening test for lung cancer is low-dose computed tomography, which is time-consuming and costly. A minimally invasive approach could be used to accelerate earlier detection of lung cancer.
miRNAs and lncRNAs contribute to the post-transcriptional regulation of mRNAs in all cell types for both healthy and diseased cells. Various miRNAs and lncRNAs have also been identified as either oncogenes or tumor suppressors. As both miRNAs and lncRNAs exhibit stable expression in circulation, their potential as tumor biomarkers is promising. To unlock potential miRNA and lncRNA signatures in circulation, high-performance tools capable of detecting low copy numbers and rare transcripts are imperative. For miRNA expression profiling, the miScript PCR System offers a suite of specialized products designed to overcome common challenges associated with circulating miRNA quantification. Similarly, the human RT² lncRNA PCR Arrays offer a leading solution for circulating lncRNA quantification. These technologies will enable cutting-edge discoveries that identify minimally invasive biomarker signatures for the early detection of lung cancer.
Materials and Methods
Serum from NSCLC patients and healthy donors (Bioreclamation, NY, and Asterand, Detroit, MI) was analyzed. The control group comprised total RNA from healthy donor serum samples. Group 1 comprised total RNA from NSCLC cancer patient serum samples. Total RNA from 200 µl serum was purified with the miRNeasy Serum/Plasma Kit.
For miRNA quantification, the miScript II RT Kit was used for cDNA synthesis. The Human Serum/Plasma miScript miRNA PCR Array was used for miRNA detection with the miScript SYBR Green PCR Kit. For lncRNA detection, The RT² PreAMP cDNA Kit and RT² Human lncRNA Cancer PathwayFinder PreAMP Primer Mix were used for cDNA synthesis and target lncRNA preamplification. The Human RT² lncRNA Cancer PathwayFinder PCR Array was used for lncRNA detection with the RT² SYBR Green qPCR Mastermix. For both miRNA and lncRNA, real-time PCR was carried out on an Applied Biosystems 7900HT Sequence Detection System. Data was analyzed using the GeneGlobe Data Analysis Center.
Regarding miRNA quantification, the Human Serum/Plasma miScript miRNA PCR Array interrogated the expression of the 372 most abundantly expressed miRNAs in serum or plasma. When ±3-fold was used as a cutoff for expression regulation, 65 miRNAs exhibited differential expression. Upon applying a p-value of 0.05, the increased expression of 12 miRNAs and the decreased expression of one miRNA were determined to be significant (Figure 1). Differentially expressed miRNAs include hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-140-3p, hsa-miR-143-3p, hsa-miR-145-5p, and hsa-miR-338-3p that have been previously associated with lung cancer.
For lncRNA detection, the Human RT² lncRNA Cancer PathwayFinder PCR Array enabled the detection of signal from 41 cancer-related lncRNAs in NSCLC patient serum samples – thus, the RT² lncRNA Cancer PathwayFinder PCR Array and PreAMP Kit simplifies lncRNA detection in serum. In contrast to healthy donor serum control samples, there was a trend for increased levels of lncRNA in cancer patient serum samples. Among these increased lncRNAs in cancer patients, two, PVT1 and RMRP, were significantly upregulated (77-fold and 24-fold, p<0.01, Cq<25 in cancer samples) (Figure 2). These significant changes in the serum of NSCLC cancer patients, demonstrated the possibility of using lncRNAs as lung cancer biomarkers.
Figure 1. Differential detection of miRNA expression using the miScript PCR System. Volcano plot of miRNA expression changes in NSCLC patient serum samples, compared with healthy donor serum samples. Y: p-value; X: log2 (fold change).
Figure 2. Successful detection of lncRNAs in serum samples with preamplification. Volcano plot of lncRNA gene expression changes in NSCLC patient serum samples, compared with healthy donor serum samples. Y: p-value; X: log2 (fold change).
The miScript PCR System enables robust quantification of serum miRNAs.
RT2 lncRNA PCR Arrays simplify lncRNA profiling in serum samples.
With preamplification, lncRNA expression changes in serum can be identified.
miScript miRNA PCR Arrays and RT2 lncRNA PCR Arrays can help identify differentially expressed miRNAs and lncRNAs in cancer serum samples compared with normal controls, providing the opportunity to identify novel biomarkers.