miRCURY LNA miRNA Focus PCR Panels

Serum/Plasma miRNA PCR Panels

Why study miRNAs in serum and plasma?
miRNAs are present in small amounts in serum and plasma. They circulate in the blood in a highly stable, cell-free form, suggesting that they are protected from the RNA-degrading enzymes found in blood (see figures miRNAs are stable in serum and plasma and Complete miRNA serum profile).

miRNA profiles have been shown to be altered in serum and plasma taken from patients suffering from cancer and other diseases. This makes miRNAs promising candidates for use in non-invasive discovery of important new biomarkers for a wide range of diseases and biological processes. Blood samples are easy to obtain and carry little risk to the patient, compared with biopsies and surgical procedures.

Challenges of miRNA profiling in serum and plasma
Serum and plasma only contain a fraction of the RNA found in whole blood (see figure Blood serum and plasma contain small amounts of RNA). Most of the RNA is found in the buffy coat, which mainly consists of white blood cells and platelets. To get accurate miRNA profiles, it is important to ensure that the serum/plasma is not contaminated by RNA from blood cells, as this would skew the results of the experiment.

Several other challenges exist when profiling miRNAs in serum and plasma, including:

• Small amounts of RNA prevent use of standard RNA quality control methods
• RNA from serum/plasma contains inhibitors of PCR enzymes
• Standard reference genes, such as U6, are not present in serum and plasma, making it difficult to choose good reference genes

Overcoming these challenges requires an extremely accurate miRNA qPCR system. The miRCURY LNA miRNA PCR system has a proven track record for reliable detection of miRNAs in serum and plasma samples.

Cancer Focus miRNA PCR Panels

miRNA and cancer
Numerous studies have demonstrated links between miRNA dysregulation and various disease states, including a diverse array of human cancers. miRNAs have been shown to act as both oncogenes and tumor suppressors. The high relative stability of miRNA in common clinical source materials (e.g., FFPE blocks, plasma, urine, saliva, etc.) and the ability of miRNAs to function as molecular markers for discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of applications. miRNAs can be used as disease classifiers for different types of cancers or as biomarkers to monitor the effects of therapy on cancer prognosis and progression.

Identify miRNA biomarkers or new classifiers
Cancer Focus miRNA PCR Panels can be used for many types of cancer miRNA profiling from samples including FFPE and biofluids. These miRNA profiles can be used to identify unique cancer signatures and help identify biomarkers or classifiers for cancer types and progression (see figures Six types of cancers compared and miRNA expression from four types of cancer).

Exosome Focus miRNA PCR Panels

How the assays were selected
All miRNA assays in the Exosome Focus miRNA PCR Panels were selected based on a combination of experimental data from in-house studies on exosomes using our miRCURY LNA miRNA PCR System and results published in peer-reviewed journals. The assays are not a complete list targeting all miRNAs found in exosomes, but they target those miRNAs typically found (see figure Urine and CSF miRNA profiles). If missing some miRNAs is a concern, we recommend running a full miRNome study using the miRCURY LNA miRNA miRNome PCR Panels.

Serum/Plasma miRNA PCR Panels

Product coverage
All 179 assays on the Serum/Plasma miRNA PCR Panels have been carefully selected based on our vast number of in-house analyses of miRNA expression in blood, serum and plasma samples, as well as on the limited number of peer-reviewed published papers available. For a complete list of miRNA assays on the panel, refer to the plate layout file.

Over 1 million in-house and collaborative data points from serum/plasma samples collected from healthy and diseased individuals have been used in the selection of relevant miRNAs for the panel. This includes miRNA expression data from different stages of various cancers, neurological disorders, neurodegenerative diseases, allergies, inflammation, etc. Our database is largely based on total miRNA extracted from serum/plasma, so a particular circulating miRNA should be on the focus panel, regardless of whether it is preferentially protein bound, found in exosomes, microvesicles or otherwise compartmentalized. This results in a very comprehensive panel with broad applications.

The Serum/Plasma miRNA PCR Panels are available in two formats:

• 96 well, ready-to-use: two 96-well PCR plates with a total of 179 LNA miRNA primer sets commonly found in human serum/plasma, including 7 candidate reference miRNAs; compatible with most real-time PCR instruments (see table below).
  The product to order consists of 4 panel sets (4x two 96-well plates).
• 384 well, ready-to-use: one 384-well PCR plate with two replicates of 179 LNA miRNA primer sets commonly found in human serum/plasma, including 7 candidate reference miRNAs; compatible with most real-time PCR instruments (see table below).
  The product to order consists of 4 panel sets (2x one 384-well plates).

The plates are produced in ready-to-use format, which means that the primer sets are dispensed and lyophilized in the well in an amount sufficient for one 10 µl reaction per well.

Control assays
Finding good reference genes in serum and plasma samples can be challenging. Some of the common house-keeping genes, such as U6 are not present in these samples, and the overall RNA concentration is low. The Serum/Plasma Focus miRNA PCR Panels contain 7 potential reference genes:

• miR-103-3p, miR-191-5p and miR-423-5p: these are potential reference genes found on our miRNome PCR Panels and can be used by researchers migrating to the focus panels
• miR-93-5p and miR-425-5p: these potential reference genes are chosen because they are usually stably expressed in serum/plasma
• miR-451a and miR-23a-3p: these can be used as a control for hemolysis. If ∆C_q(miR-23a-3p – miR-451a) is >7, it may be an indication of contamination of the serum/plasma samples.

In addition, each panel also contains qPCR assays for the 5 synthetic RNAs in the RNA Spike-in kit (cel-miR-39-3p, UniSp2, UniSp4, UniSp5 and UniSp6) that may be used for monitoring the cDNA synthesis or RNA purification (UniSP6 is also provided in the miRCURY LNA RT Kit). Finally, each panel contains an inter-plate calibrator in triplicate and an empty negative control. When analyzing qPCR data, we recommend normalizing across the entire panel for best results.

Cancer Focus miRNA PCR Panels

The miRCURY LNA miRNA Cancer Focus PCR Panel targets 84 human miRNAs related to cancer that have been shown to be important for tumorigenesis and in cancer diagnostics. These miRNAs can be used as markers for 27 different types of cancer. They have been identified from a large biobank containing thousands of FFPE samples, as well as from literature data. For a list of references of each miRNA on the panel, refer to the plate layout file.

Product coverage
The Cancer Focus miRNA PCR Panel is available in:

• 96-well plate format: one Cancer Focus Panel on one 96-well plate.
  The product to order consists of 4 panel sets (4x one 96-well plates).
• 384-well plate format: eight Cancer Focus Panels delivered in two 384-well plates (sufficient for 8 samples). These plates have been specifically designed for fast and easy manual and multi-pipetting.
  The product to order consists of 8 panel sets (2x one 384-well plates).

Both formats are ready-to-use with 10 µl reactions per well.

The panel contains assays for the following:

• 84 miRNAs specifically linked to cancer
• 3 miRNA reference genes: miR-103-3p, miR-191-5p, miR-423-5p
• snRNA reference genes: SNORD49A, U6 snRNA, SNORD38B
• 3 Inter-plate calibrators (UniSp3 IPC)
• 5 RNA spike-ins (cel-miR-39-3p, UniSp2, UnuSp4, UniSp5 and UniSp6)

The Cancer Focus miRNA PCR Panels are compatible with most real-time PCR instruments (see table below).

Exosome Focus miRNA PCR Panels

Key benefits of the Exosome Focus miRNA PCR Panels include:

• All typically expressed miRNAs found in urine or cerebrospinal fluid samples in one PCR panel
• A highly flexible solution that allows you to focus on the miRNAs relevant to your exosome project
• Intuitive plate designer allows replacement of assays in the plates before ordering
• Sensitivity and specificity for accurate miRNA profiling
• Fast and easy, ready-to-use format enables direct sample application and a full profile in less than 3 hours
• High compatibility 96- and 384-well formats are compatible with most real-time PCR instruments

Product coverage
The Exosome Focus miRNA PCR Panels are offered as pre-defined Custom miRNA PCR Panels. This includes pre-defined lists of assays for miRNAs expressed in either exosomes from urine or exosomes from cerebrospinal fluid (CSF). Both groups of assays are designed to fit into a single 96-well PCR plate or as 4 replicates in a 384-well PCR plate.

The Urine Exosome Focus miRNA PCR Panel contains:

• 87 miRNA LNA PCR primer sets targeting human miRNAs expressed in human exosomes from urine
• 5 of the miRNA assays can also be used as potential internal control primers: hsa-miR-103-3p, hsa-miR-191-5p, hsa-miR-93-5p, hsa-miR-423-3p and hsa-miR-423-5p
• 5 RNA spike-in controls available for selection in the panel configurator for use as positive controls (e.g., for quality control of the RNA purification and the RT reaction)
• 3 inter-plate calibrators (3 x UniSp3 IPC)

The CSF Exosome Focus miRNA PCR Panel contains:

• 87 miRNA LNA PCR primer sets targeting human miRNAs expressed in human exosomes from cerebrospinal fluid
• 3 of the miRNA assays can also be used as potential internal control primers: hsa-miR-103-3p, hsa-miR-191-5p and hsa-miR-93-5p
• 5 RNA spike-in controls available for selection in the configurator for use as positive controls (e.g., for quality control of the RNA purification and the RT reaction)
• 3 inter-plate calibrators (3 x UniSp3 IPC)

The easy-to-follow, 3-hour protocol saves you both time and effort in the laboratory. Simply dilute the synthesized cDNA, combine it with the miRCURY LNA SYBR Green Master Mix and aliquot into the 96- or 384-well PCR plate containing the pre-aliquoted PCR Panel. The plate is now ready to run in your real-time PCR instrument.

miRNA quantification in serum, plasma, exosomes, urine and other liquid biopsies; biomarker discovery