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Kansas State University

Fluoroskan Ascent FL

 

Fluoroskan Ascent FL


Application: Total RNA Molecular Probes Rediplate 96 Ribogreen Assay

Generic Description of how to use the machine and the software

Total RNA Molecular Probes Rediplate 96 Ribogreen Assay:

Introduction:

The RediPlateTM 96 RiboGreen« RNA Assay Kit is a rapid, simple and highly sensitive fluorescence-based microplate assay for quantitating RNA. The kit uses RiboGreen reagent, a nucleic acid stain that shows bright green fluorescence upon binding to RNA or DNA. Eleven of the strips (88 wells) are preloaded with the RiboGreen reagent. The remaining strip, marked with blackened tabs, contains a series of RNA standards for generating standard curves.

Applications:

The RiboGreen Assay is ideal for measuring yields of RNA preparations for

  1. Microarray experiments

  2. Reverse transcription PCR (RT-PCR)

  3. Differential display PCR

  4. Northern blot analysis

  5. S1 nuclease assays

  6. cRNA library preparation.

Kit Contents:

• RediPlate RiboGreen RNA assay microplate
• RediPlate TE buffer

Note: The Kit should be kept at -20˚C or below and should be prevented from RNase contamination.

Experimental Protocol ( for the range of 15-1000 ng/mL ):

  1. Allow the kit components to warm to room temperature.
    Note: Do not open the foil packet until it is warm. It will take about 20 min.


  2. Thaw the TE buffer to room temperature which may take more than 20 min. The TE buffer can be placed in a warm water bath to accelerate thawing.
    Note: The buffer may be stored at 2-6˚C for convenience after thawing.


  3. Determine the number of strips required and carefully cut through the self-adhesive sealing film using a razor blade. Store the extra strips at -20˚C.


  4. Preparation of standard curve samples:- Add 100ÁL of RediPlate TE buffer to each well in column 1 (with blackened tabs). Mix well, wait for 5 min to rehydrate RNA completely and mix again.

  5. RNA standards provided in column one of the microplate.

    Well Mass of dried RNA standard in well Concentration of reconstituted RNA standard Concentration of RNA standard in assay
    A 1000 ng 10,000 ng/mL 1000 ng/mL
    B 500 ng 5000 ng/mL 500 ng/mL
    C 250 ng 2500 ng/mL 250 ng/mL
    D 100 ng 1000 ng/mL 100 ng/mL
    E 40 ng 400 ng/mL 40 ng/mL
    F 15 ng 150 ng/mL 15 ng/mL
    G 5 ng 50 ng/mL 5 ng/mL
    H 0 ng 0 ng/mL 0 ng/mL

  6. Add 180ÁL of TE buffer to all of the wells that are required for all experimental samples and standard curve samples.

  7. Pipette 20ÁL of RNA from each of the standard RNA wells to the corresponding assay wells. Mix well.

  8. Add 5-20ÁL of the RNA samples to the assay wells. Mix well.
    Note:

    1. The final volume in each well should be 200ÁL. So, it has to be adjusted with TE buffer accordingly.

    2.  
    3. In order for the RediPlate 96 RiboGreen Assay to be accurate, the amount of RNA in the assay well must fall within the linear range of the assay (~15-1000 ng/mL or ~3-200 ng in the 200ÁL assay volume).
       

  9. Incubate the samples 10 min at room temperature, protected from light.

  10. Read the fluorescence using Fluoroskan Ascent Machine, as described below.

To read fluorescence:

  • Turn on the instrument

  • Start the software by clicking the icon on the desktop

Ascent Icon on Desktop

 



 

  Open an existing/new application

PROCEDURE DESKTOP:

TOP

When the software is opened, the user can view two tabs:

  • Procedure

  • Result

The Parameters, Area definition, Layout and Settings are specified in the Procedure tab which is named in the software as “Procedure Desktop”. The highly visual Procedure desktop is used for instrument control and assay setup.

By clicking ‘New’ the Window ‘New session’ pops up with the menu of all the templates.

Procedure Desktop

Open the session file RNAAssay.sec to carry out a fluoroscence based microplate assay for quantitating RNA.
The Procedure Toolbar contains the following buttons:

Procedure Toolbar:

Procedure Toolbar

The Procedure Desktop shows the tabs Parameters, Area Definition, Layout, Settings and RunStatus.
 

Procedure Desktop

AREA DEFINITION:

TOP

The area definition is used to select the target areas for the Measure and Dispense steps. For this application, we use the wells A, B, C of the 5th Column (which is highlighted in yellow color when selected). 


Area Definition

 
LAYOUT:

TOP

The Layout function allows the user to specify the wells. The plate is described in the window. The data fields are:

Type: You can select the type of an item from the drop-down list. This can be either CALIBR., CONTROL, SAMPLE, or BLANK.

The selected types can be applied or removed using the Apply and Remove button.

Layout function

Parameters:

In the parameters tab , click on the Measure Button. A Window pops up which has the stepname, unit, steptime, measurement method, measurement type, integration time and the lag time.

Note: In the filter pair, set the excitation to 485 and the emission to 527 for RiboGreen.

Parameters

SETTINGS:

TOP

SETTINGS

RUNSTATUS:

TOP

RUNSTATUS

The yellow color in the wells is changed to red to indicate the wells being read for fluorescence.

The Done Status bar indicates the level of proceeding of the process.

The START button initiates up the quantitation process. After the RunStatus is done, the result desktop pops up.

RESULT DESKTOP:

TOP

The tab which shows the values obtained from fluorescence is called the “Result Desktop”. The extremely flexible Result Desktop is built as a familiar spreadsheet environment. In addition to the comprehensive built in automatic calculations, the function tool can be used to perform basically any custom-made calculations.
 

RESULT DESKTOP


Output data:

The values from the result desktop can be copied and pasted into Excel Spreadsheet (Note: A shortcut to Excel has been placed on the desktop).