High sensitivity ( low input ) miRNA Illumina library preparation protocol by TailorMix miRNA Sample Preparation Kit
Thaw Mix C300 from - 20°C storage .
Allow it to equilibrate to room temperature for a minimum of 30 minutes before use .
Pre - heat the thermal cycler to 70°C and pre - heat another thermal cycler to 25°C if available .
Denature the RNA Sample by assembling the following components in a sterile 200 μL PCR tube on ice : RNA Sample , 6μL ; Mix A300 2μL ;
Vortex mix thoroughly and incubate at 70°C for 1 minute and then place the tube on ice .
Set up the following 3’ Adapter Ligation reaction on ice : Denatured RNA mix from step 4 , 8μL ; Mix B300 , 2μL ; Mix C300 , 6.5μL ;
Vortex mix thoroughly and pulse spin .
Incubate at 25°C for 1 hour .
Vortex the TailorMag Purification Beads ( TPB ) until they are evenly suspended .
Prepare 80 % ethanol for rinse step .
Add 30 μL of TPB with each 3’ - adapter ligated sample from Step 6 .
Vortex mix thoroughly and pulse spin .
Incubate at room temperature for 15 minutes the following : 3’ - adapter ligated sample from Step 6 , 16.5μL ; TailorMag Purification Beads ( TPB ) , 30μL ;
Place the sample tube on the magnetic stand at room temperature for 5 minutes .
Carefully remove and discard 40 μL of the supernatant .
Keep sample tube on the magnetic stand .
Gently rinse the TPB pellet with 150 μL of 80 % ethanol without disrupting the TPB pellet .
Discard the rinse solution .
Air dry sample tube at room temperature .
Remove sample tube from the magnetic stand .
Add 7 μL of nuclease free water to the dried TPB pellet .
Vortex to resuspend and pulse spin .
Incubate sample resuspension at room temperature for 2 minutes .
Set up the following 5’ Adapter Ligation reaction on ice : 3’ Adapter Ligated RNA from step 18 , 7μL ; Mix D300 , 3μL ; Mix E300 , 2μL ;
Gently pipette mix thoroughly and incubate at 25°C for 1 hour and then place the tube on ice .
Pre - heat the thermal cycler to 50°C .
Set up the following cDNA Synthesis reaction on ice : 3’ and 5’ Adapter Ligated RNA from Step 16 ( contains TPB ) , 12μL ; Mix F300 , 2μL ; Mix G300 1μL ;
Vortex mix thoroughly and pulse spin .
Incubate at 50°C for 1 hour and then place the tube on ice .
Set up the following PCR reaction in a fresh sterile 200 µl PCR tube on ice : First strand cDNA from Step 19 ( contains TPB ) , 5μL ; Mix H300 , 18μL PCR Primer 1μL Index Primer * , 1μL ;
Only one of the Index primers is used for each sample .
Vortex mix thoroughly and pulse spin .
Amplify the samples in the thermal cycler using the following PCR cycling conditions : 98°C for 30 seconds ; 15 cycles of : 98°C for 5 seconds ; 60°C for 15 seconds ; 72°C for 1 minute ; 72°C for 5 minutes .
Hold at 4°C .
PCR yield can be monitored by running an Agilent BioAnalyzer High Sensitivity DNA assay using a dilution of 1 μL of PCR product and 9 μL of nuclease - free water .
A typical result shows a distinct peak at approximately 140bp ( Figure 2 in Guidelines ) .
Determine the volume of TBE buffer needed and dilute 5X TBE Buffer to 1X for use in gel electrophoresis .
Assemble the gel electrophoresis apparatus .
Mix 2 μL of Custom Ladder with 2 μL of Hi - Density TBE Sample Buffer .
( Optional ) Mix 1 μL of 100bp DNA ladder with 1 μL of Hi - Density TBE Sample Buffer .
Add 2.5 μL of Hi - Density TBE Sample Buffer to 25 μL of PCR product and pipette mix thoroughly .
Load 25 μL of the PCR product - Sample Buffer mix into one well in the middle of the 8 % PAGE gel .
Refer to Figure 3 in Guidelines for an example .
Load 2 μL of the custom ladder and dye mix into the neighboring wells of the PCR products .
( Optional ) Load 2 μL of the 100bp DNA ladder and dye mix into a separate well .
Run the gel for 75 minutes at 145V and immediately remove the gel from the apparatus .
Prepare TE buffer with 0.1 % Tween - 20 : TE buffer , 9,990μL ; Tween - 20 10μL ;
Open the gel cassette and stain with 1μg / mL ethidium bromide solution according to the manufacturer’s instructions .
Place the gel on a UV Transilluminator and observe the banding pattern ( Figure 3 in Guidelines ) .
( Alternative ) Stain gel with Sybr Gold according to the manufacturer’s instructions and observe the banding pattern on a Dark Reader Transilluminator .
Place the gel breaker tube into a sterile 1.5mL microcentrifuge tube .
The 140bp band represents the highest concentration of micro RNA library .
To excise the 140bp band , align the center of the gel cutter tool with the 140 bp band of the custom ladder ( Figure 4 in Guidelines ) .
Press down firmly into the gel and excise the gel fragment .
Insert the gel cutter tool containing the gel slice into the gel breaker tube .
Pulse - spin the gel cutter and gel breaker assembly in a minifuge .
Make sure the gel slice is collected in the gel breaker tube .
Remove gel cutter from the assembly and discard .
Add 30 μL of TE buffer with 0.1 % Tween - 20 to the gel breaker tube containing the gel slice .
Centrifuge the gel breaker assembly in a bench top centrifuge at maximum speed ( approximately 13,000x G ) for two minutes at room temperature .
Ensure that all of the gel has moved through the holes into the collection tube .
Elute the micro RNA library by shaking the tube at 600 rpm at room temperature overnight .
To collect the micro RNA library , spin the gel mix at maximum speed ( approximately 13,000x G ) for 2 minutes .
With a P10 pipette , gently transfer 20 - 25 µl of eluate from gel mix to a fresh 1.5ml tube .
Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysis of your sample library .
Use 1 μL of resuspended construct from step 47 on a High Sensitivity DNA chip to check the size , purity and concentration of the sample .
