Cat. #P-7102
Intro

SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric)

Base Catalog # P-7102
PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE

Uses

The SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric) is suitable for detecting N6-methyladenosine (m6A) RNA methylation status directly using total RNA isolated from any species such as mammals, plants, fungi, bacteria, and viruses.

Input RNA

The amount of RNA for each assay can be 100 ng to 300 ng. For optimal quantification, the input RNA amount should be 200 ng, as the abundance of m6A is generally less than 0.1% of total RNA.

Internal Control

Both negative and positive RNA controls are provided in this kit. A standard curve can be performed (range: 0.02 to 1 ng of m6A) or a single quantity of m6A can be used as a positive control. Because m6A content can vary from tissue to tissue, and from normal and diseased states, or vary under treated and untreated conditions, it is advised to run replicate samples to ensure that the signal generated is validated. This kit will allow the user to quantify an absolute amount of m6A and determine the relative m6A RNA methylation states of two different RNA samples.

Precautions

To avoid cross-contamination, carefully pipette the sample or solution into the strip-wells. Use aerosol-barrier pipette tips and always change pipette tips between liquid transfers. Wear gloves throughout the entire procedure. In case of contact between gloves and sample, change gloves immediately.

Kit setup

Kit Contents & Storage

Component96 Reactions
Cat. #P-7102-96		Storage Upon Receipt
WB (10X Wash Buffer)14 ml4°C
HBS (High Binding Solution)10 mlRT
NC (Negative Control, 100 µg/ml)*20 µl-20°C
PC (Positive Control, m6A 2 µg/ml)*20 µl-20°C
m6Ab (m6A Antibody, 1000X)*10 µl4°C
MSI (Signal Indicator, 1000X)*10 µl-20°C
ES (Enhancer Solution, 1000X)*10 µl-20°C
DS (Developer Solution)10 ml4°C
SS (Stop Solution)10 mlRT
8-Well Assay Strips (With Frame)124°C

* Spin the solution down to the bottom prior to use.

Take Note!

The NC (Negative Control) is an RNA containing no m6A. The PC (Positive Control) is m6A oligos and is normalized to have 100% of m6A.

Shipping & Storage

The kit is shipped in two parts: the first part at ambient room temperature and the second part on frozen ice packs to maintain approximately 4°C.

Upon receipt: (1) Store NC, PC, MSI and ES at -20°C away from light; (2) store WB, m6Ab, DS, and 8-Well Assay Strips at 4°C away from light; (3) Store HBS and SS at room temperature away from light.

All components of the kit are stable for 6 months from the date of shipment, when stored properly.

Take Note!

Check if WB contains salt precipitates before use. If so, briefly warm at room temperature or 37°C and shake the buffer until the salts are re-dissolved.

Before you begin

Materials Required But Not Supplied

Assay principle

Principle & Procedure

The SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric) contains all reagents necessary for the quantification of m6A in RNA. In this assay, total RNA is bound to strip wells using high binding solution. m6A is detected using m6A Detection Complex. The detected signal is enhanced and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The amount of m6A is proportional to the OD intensity measured.

Schematic procedure for the SpeedRun One-Hour m6A RNA ELISA Kit
Fig. 1. Schematic procedure for SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric).
Assay protocol

Starting Materials

For the best results, please read the protocol in its entirety prior to starting your experiment.

Input RNA Amount

Total RNA amount can range from 100 ng to 300 ng per reaction. An optimal amount is 200 ng per reaction. Starting RNA may be in water or in a buffer such as TE. You can use your method of choice for RNA isolation.

RNA Storage

Isolated total RNA can be stored at -20°C (short term) or -80°C (long term) until use.

Illustrated m6A positive control standard curve
Fig. 2. Illustrated standard curve generated with the m6A positive control. Actual results may vary.
Step 1

Working Buffer and Solution Preparation

Prepare Diluted WB (1X Wash Buffer) by adding 12 ml of WB (10X Wash Buffer) to 108 ml of distilled water.

Take Note!

This Diluted WB can now be stored at 4°C for up to six months.

The anticipated approximate volumes of reagents needed are reflected below for this assay.

Reagents1 well8 wells
(1 strip)		16 wells
(2 strips)		48 wells
(6 strips)		96 wells
(12 strips)
Diluted WB1 ml8 ml16 ml48 ml96 ml
HBS100 µl800 µl1600 µl4800 µl9600 µl
m6A Detection Complex50 µl400 µl800 µl2400 µl4800 µl
DS0.1 ml0.8 ml1.6 ml4.8 ml9.6 ml
SS0.1 ml0.8 ml1.6 ml4.8 ml9.6 ml
NCN/A4 µl4 µl4 µl4-8 µl
PCN/AN/AOptional6 µl6-12 µl
Step 2

Standard or Positive Control (PC) Curve Preparation

Preparation of Diluted Positive Control

Single Point Control Prep

Dilute PC (Positive Control) with 1X TE to 0.5 ng/µl (1 µl PC + 3 µl TE).

Suggested Standard Curve Prep

First, dilute PC to 0.5 ng/µl (ex: 3 µl of PC + 9 µl of 1X TE). Then, further prepare 6 different concentrations with the 0.5 ng/µl PC and 1X TE into 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 ng/µl according to the following dilution chart:

TubePC (0.5 ng/µl)1X TEResulting PC Concentration
11.0 µl49.0 µl0.01 ng/µl
21.0 µl24.0 µl0.02 ng/µl
31.0 µl9.0 µl0.05 ng/µl
41.0 µl4.0 µl0.1 ng/µl
52.0 µl3.0 µl0.2 ng/µl
64.5 µl0.0 µl0.5 ng/µl
Take Note!

1) The above volumes will be sufficient for one standard curve in duplicate (12 wells total excluding NC). The PC concentrations are based on per assay well, not per microliter; 2) Keep each of diluted solutions, except Diluted WB (1X Wash Buffer), on ice until use. Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day.

Step 3

RNA Binding Strip Well Setup

Review the configuration of the strip-well plate setup for standard curve preparation strips, showing strip 1 to 6 (7 through 12 can be configured as Sample). The controls and samples can be measured in duplicate, loaded vertically instead of horizontally.

Table 1. The suggested strip-well plate setup using a single point positive control, showing strips 1 to 6. Strips 7 to 12 can be configured as Sample. The controls and samples can be measured in duplicate.

Well #Strip 1Strip 2Strip 3Strip 4Strip 5Strip 6
ANCSampleSampleSampleSampleSample
BNCSampleSampleSampleSampleSample
CPCSampleSampleSampleSampleSample
DPCSampleSampleSampleSampleSample
ESampleSampleSampleSampleSampleSample
FSampleSampleSampleSampleSampleSample
GSampleSampleSampleSampleSampleSample
HSampleSampleSampleSampleSampleSample

Table 2. The suggested strip-well plate setup for standard curve preparation

Well #Strip 1Strip 2Strip 3Strip 4Strip 5Strip 6
ANCPC 0.2 ng/wellSampleSampleSampleSample
BNCPC 0.2 ng/wellSampleSampleSampleSample
CPC 0.02 ng/wellPC 0.4 ng/wellSampleSampleSampleSample
DPC 0.02 ng/wellPC 0.4 ng/wellSampleSampleSampleSample
EPC 0.04 ng/wellPC 1 ng/wellSampleSampleSampleSample
FPC 0.04 ng/wellPC 1 ng/wellSampleSampleSampleSample
GPC 0.1 ng/wellSampleSampleSampleSampleSample
HPC 0.1 ng/wellSampleSampleSampleSampleSample
Step 4

RNA Binding

a
Predetermine the number of strip wells required for your experiment. Carefully remove un-needed strip wells from the plate frame and place them back in the bag (seal the bag tightly and store at 4°C).
b
Add 80 µl of HBS (High Binding Solution) to each well.
c
Negative Control Wells: Add 2 µl of NC to each NC well.
d
Standard Wells: Add 2 µl of Diluted PC (m6A Positive Control) to each standard well (see the designated wells depicted in the Table under "RNA Binding Strip Well Setup" above).
e
Sample Wells: Add 2 to 4 µl of RNA sample (200 ng) to each sample well.
Take Note:

(1) For a single point control, add 2 µl of PC at a concentration of 0.5 ng/µl to each PC well as shown in Table 1; (2) For the standard curve, add 2 µl of Diluted PC at concentrations of 0.01 to 0.5 ng/µl (see the chart in Step 2). The final amounts should be 0.02, 0.04, 0.1, 0.2, 0.4 and 1 ng per well. (3) For optimal binding, sample RNA volume added should not exceed 8 µl. (4) To ensure that NC, Diluted PC, and sample RNA are completely added into the wells, the pipette tip should be placed into the HBS solution in the well and aspirated in/out 1-2 times.

Mix solution by gently tilting from side to side or shaking the plate several times.

f
Tightly cover strip plate with parafilm M or plate seal to avoid evaporation and incubate at 37°C for 25 minutes.
g
During the last 10 minutes of sample incubation, prepare the m6A Detection Complex Solution: In each 1 ml of Diluted WB add 1 µl of m6Ab, mix and then add 1 µl of MSI (Signal Indicator) and 1 µl of ES (Enhancer Solution). Mix well.
h
Remove the reaction solution from each well. Wash each well one time with 150 µl of the Diluted WB (1X Wash Buffer).
Step 5

m6A Level Detection and Signal Measurement

a
Add 50 µl of the m6A Detection Complex Solution to each well, then cover and incubate at room temperature for 25 minutes.
b
Remove the m6A Detection Complex Solution from each well.
c
Wash each well with 150 µl of the Diluted WB (1X Wash Buffer) each time for four times.
d
Add 100 µl of DS (Developer Solution) to each well in a column, not row, simultaneously in a vertical fashion with a multi-channel pipette so that replicates are developed at the same time. Gently shake the plate against a flat surface for 5-10 seconds and incubate at room temperature for 3-4 minutes. Monitor color development in the sample wells and control wells. After a few minutes, the DS (Developer Solution) will turn blue in the presence of sufficiently bound m6A RNA. The color in the NC wells will remain generally unchanged or slightly changed.
e
When the color in the 1 ng PC wells turns deep blue, stop the enzyme reaction by adding 100 µl of SS (Stop Solution) to each well in the same order as Step 5d. Mix the solution by gently shaking the plate against a flat surface and wait 3-4 minutes to allow the color reaction to be completely stopped. The color will change to yellow after adding SS (Stop Solution) and the absorbance should be read on a microplate reader at 450 nm within 2-15 minutes.
Take Note!

The color development time may vary from 1-10 minutes based on the speed of color change, but is typically 3-4 minutes.

Step 6

m6A Calculation

Relative Quantification

To determine the relative m6A RNA methylation status of two different RNA samples, a simple calculation for the percentage of m6A in your total RNA can be carried out using the following formula:

m6A % =(Sample OD - NC OD) ÷ S(PC OD - NC OD) ÷ Px 100%

S is the amount of input sample RNA in ng.
P is the amount of input positive control (PC) in ng.

Absolute Quantification

To quantify the absolute amount of m6A using an accurate calculation, first generate a standard curve and plot the OD values (background (NC)-subtracted) versus the amount of PC at each concentration point. Next, determine the slope (OD/ng) of the standard curve using linear regression (Microsoft Excel's linear regression functions are suitable for such calculation). Use the most linear part of the standard curve (at least 4 concentration points, including NC point) for optimal slope calculation. Now calculate the amount and percentage of m6A in your total RNA using the following formulas:

m6A (ng) =Sample OD - NC ODSlope
m6A % =m6A Amount (ng)Sx 100%

S is the amount of input sample RNA in ng.

Take Note!

In the event that the standard curve is flat due to high ODs starting from the lowest amount of PC or is flat at high amount of PCs because of a saturated signal intensity due to extended color development time, the m6A percentage can be calculated with logarithmic or polynomial second order regression (see the Appendix section).

A Brief Overview

N6-methyl-adenosine (m6A) is the most common and abundant modification on RNA molecules present in eukaryotes. The m6A modification is catalyzed by a methyltransferase complex METTL3 and removed by the recently discovered m6A RNA demethylases FTO and ALKBH5, which catalyze m6A demethylation in an α-ketoglutarate (α-KG)- and Fe2+-dependent manner

m6A accounts for more than 80% of all RNA base methylations and exists in various species. m6A is mainly distributed in mRNA and also occurs in non-coding RNA such as tRNA, rRNA and snRNA. The relative abundance of m6A in mRNA transcripts has been shown to affect RNA metabolism processes such as splicing, nuclear export, translation ability and stability and RNA transcription. Abnormal m6A methylation levels induced by defects in m6A RNA methylase and demethylase could lead to dysfunction of RNA and cause disease

Reversible m6A methylation schematic
Reversible m6A methylation in mRNA (Niu et al, 2013)

To address this, EpigenTek offers a series of m6A RNA quantification kits to quantify m6A and further refines its m6A RNA assay expertise with the development of the SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric). This kit has the following advantages and features:

  • Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed in 1 hour.*
  • High sensitivity, of which the detection limit can be as low as 10 pg of m6A.
  • Unique binding solution allows that RNA >70 nts can be tightly bound to the wells, which enables quantification of m6A from both mRNA and nc-RNA such as tRNA, rRNA and snRNA.
  • Optimized antibody and enhancer solutions allow high specificity to m6A, with no cross-reactivity to unmethylated adenosine within the indicated concentration range of the sample RNA.
  • Universal positive and negative controls are included, which are suitable for quantifying m6A from any species.
  • Strip-well microplate format makes the assay flexible for manual or high throughput analysis.
  • Simple, reliable, and consistent assay conditions.

* Based on a single sample assay in duplicate

Troubleshooting
ProblemPossible CauseSuggestion
No signal in both the positive control and sample wellsReagents are added incorrectly.Check if reagents are added in the proper order and if any steps in the protocol may have been omitted by mistake.
The well is incorrectly washed before RNA binding.Ensure the well is not washed prior to adding the positive control and sample.
The bottom of the well is not completely covered by the HBS (High Binding Solution).Ensure the solution coats the bottom of the well by gently tilting from side to side or gently shaking the plate several times.
Incubation time and temperature are incorrect.Ensure the incubation time and temperature described in the protocol are followed correctly.
Insufficient input materials.Ensure that a sufficient amount of positive control and samples are added into the wells.
Incorrect absorbance reading.Check if appropriate absorbance wavelength (450 nm) is used.
Kit was not stored or handled properly.Ensure all components of the kit were stored at the appropriate temperature and the caps are tightly capped after each opening or use.
No signal or weak signal in only the positive control wellsThe PC (Positive Control) is insufficiently added to the well in Step 4d.Ensure a sufficient amount of positive control is added.
The PC (Positive Control) is degraded due to improper storage conditions.Follow the Shipping & Storage guidance in this User Guide for storage of PC (Positive Control).
High background present in the negative control wellsInsufficient washing of wells.Check if washing recommendations at each step are performed according to the protocol.
Contaminated by sample or positive controlEnsure the well is not contaminated from adding sample or positive control RNA accidentally or from using contaminated tips.
Over development of color.Decrease the development time in Step 5d before adding SS (Stop Solution) in Step 5e.
Large variation between replicate wellsHorizontal positioning of well replicates causes inconsistent delays in pipetting and loading of reagents.Follow the vertical layout example provided in Step 3. Ensure loading of reagents is also in vertical order with a multi-channel pipette, especially when adding DS (Developer Solution) and SS (Stop Solution) in Step 5.
Color reaction is not evenly stopped due to an inconsistency in pipetting time or in pipetting volume.Ensure DS (Developer Solution) and SS (Stop Solution) is added at the same time between replicates or otherwise maintains a consistent timing in between each addition of solutions. Ensure the solution in each pipette tip is equal in the multi-channel pipette. Equilibrate the pipette tip in any solutions before adding them. Ensure the solutions, especially those with small volumes (e.g., 1 µl) are completely added into the wells. Pay special attention at the steps of adding RNA samples and preparing m6A Detection Complex Solution.
Color reaction is not evenly stopped due to an inconsistent order of adding solutions.Ensure all solutions, particularly DS (Developer Solution) and SS (Stop Solution), are added in the same order each time as all other solutions.
Residue wash buffer is present in some of the wells.Ensure the wash buffer is completely removed at each wash step.
Splashing of reagents between wells.Pipette carefully against the sides of the wells to avoid splashing.
Temperature variations across the plate.Ensure plates are evenly and fully covered during incubation steps in a stable temperature environment, away from drafts.
Large variation between sample replicate wells onlySample RNA is sedimented or uneven prior to loading to wells.Mix your sample RNA sufficiently and evenly prior to loading it into wells.
m6Ab (m6A Antibody) vial appears to be empty or insufficient in volumeBuffer evaporated due to the very small volumes, resulting in a higher concentrated antibody.Add 1X PBS buffer into the m6Ab (m6A Antibody) vial until you restore the correct, intended volume according to the Kit Contents described in this User Guide. Mix and centrifuge prior to use.
Appendix

Method 1: Target Calculation Using Logarithmic Regression

Use this method when the standard curve is flat due to high ODs starting from the lowest standard concentration.

a
Plot the average OD value on the Y-axis versus the known target level of each standard point on the X-axis.
b
Graph the logarithmic curve* (also see "Example Calculation" below) and obtain the logarithmic regression equation:
Y = aln(X) + b

Here, X = target level represented on the standard curve X-axis; Y = Sample OD; a is the regression coefficient and b is the Y-intercept.

*Microsoft Excel's logarithmic regression function can be used for easy and convenient calculation.

c
Calculate the target percentage of the samples based on the following equation, derived from the above equation:
Target% = e[(Y-b)/a] ÷ S x 100%

Here, S is the amount of input sample in ng.

Example Calculation

Example logarithmic regression curve
Data was graphed using a Standard Scatter (XY) Chart in Microsoft Excel.

In the figure above, a = 0.2508, b = 1.1329, Y = 1.274. Input sample amount was 100 ng.

Target percentage of the sample was calculated accordingly:

Target% = e[(1.274-1.1329) / 0.2508] ÷ 100 x 100% = 1.75%

Method 2: Target Calculation Using Polynomial Second Order Regression

Use this method when the standard curve is flat due to a saturated signal intensity at high standard concentrations.

a
Plot the average delta OD values on the Y-axis versus the known target level of each standard point on the X-axis.
b
Graph the second order polynomial curve* (also see "Example Calculation" below) and obtain the second order polynomial regression equation:
Y = aX2 + bX

Here, X = target level represented on the standard curve X-axis; Y = (sample OD value - negative control or blank OD) represented on the standard curve Y-axis; a and b are regression coefficients.

*Microsoft Excel's polynomial regression function can be used for easy and convenient calculation.

c
Calculate the target percentage of the samples based on the following equation, derived from the above equation:
Target% =(b2 + 4aY)0.5 - b2a÷ S x 100%

Here, S is the amount of input sample in ng.

Example Calculation

Example polynomial second order regression curve
Data was graphed using a Standard Scatter (XY) Chart in Microsoft Excel.

In the figure above, a = -0.0865, b = 0.6373, Y = (0.621 - 0.060) = 0.561. Input sample amount was 100 ng.

Target percentage of the sample was calculated accordingly:

Target% =[(0.6373)2 + (4 x -0.0865 x 0.561)]0.5 - 0.63732 x -0.0865÷ 100 x 100% = 1.02%
Take Note!

If the background-corrected sample OD is less than zero or falls outside the measurable range of the standard curve, the target level should not be calculated by extrapolation. The sample should be considered below the quantifiable range or re-assayed at an appropriate dilution, as applicable.

General Product Information

Quality Control

Each lot of the SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric) is tested against predetermined specifications to ensure consistent product quality. EpigenTek guarantees the performance of all products in the manner described in our product instructions.

Product Warranty

If this product does not meet your expectations, simply contact our technical support unit or your regional distributor. We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques.

Safety

Suitable lab coat, disposable gloves, and proper eye protection are required when working with this product.

Product Updates

EpigenTek reserves the right to change or modify any product to enhance its performance and design. The information in this User Guide is subject to change at any time without notice. Be sure to use the latest User Guide for this kit which can be accessed online at www.epigentek.com/datasheet.

Usage Limitation

The SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric) is for research use only and is not intended for diagnostic or therapeutic applications.

Intellectual Property

The SpeedRun™ One-Hour m6A RNA ELISA Kit (Colorimetric) and methods of use contain proprietary technologies by EpigenTek.