Gel - Free miRNA Illumina Library Preparation Protocol by TailorMix Gel - Free miRNA Sample Preparation Kit
3’ Adapter Ligation Thaw Mix C400 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 A400 , 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 B400 , 2μL ; Mix C400 , 6.5μL .
Note : Mix C400 is a highly viscous reagent .
Handle with care and pipette slowly to ensure the correct amount of Mix C400 is dispensed for each reaction .
Vortex mix thoroughly and pulse spin .
Incubate at 25°C for 1 hour .
Ligation Product Clean Up 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 .
Votex mix thoroughly and incubate at room temperature for 15 minutes .
Note : Do NOT perform strong centrifugation because it will separate TPB from the sample .
Place the sample tube on the magnetic stand at room temperature for 5 minutes , or until solution clears up .
Carefully remove and discard 40 μL of the supernatant .
Note : Sample recovery may be affected if the TPB pellet is disrupted .
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 .
Tip : Point pipette tip towards opposite direction as the TPB pellet .
Gently pipette the 80 % ethanol up and down once , then discard the rinse solution .
Air dry sample tube at room temperature .
Note : TailorMag Purification Beads are dried within 5 to 15 minutes at room temperature .
Proceed to Step 14 when the appearance of the TPB pellet turns form glossy / shiny ( wet ) to matte ( dry ) .
Sample recovery may be affected if beads are over - dried and appear powdery .
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 .
Note : Presence of TPB does not interfere with the enzymatic reaction .
Note : To minimize the presence of the artifact products , add Mix D400 and Mix E400 to the sample in consecutive steps .
Vortex mix thoroughly and pulse spin .
Incubate at 25°C for 1 hour and then place the tube on ice .
cDNA Synthesis Pre - heat the thermal cycler to 50°C .
Note : Presence of TPB does not interfere with the enzymatic reaction .
Vortex mix thoroughly and pulse spin .
Incubate at 50°C for 1 hour and then place the tube on ice .
Safe Stopping Point : First strand cDNA could be stored at - 20°C for up to seven days .
PCR Amplification Note : This protocol has been optimized using 900 ng of purified high quality human kidney total RNA as input .
Because miRNA populations vary among different tissue types and species , the use of total RNA from other tissue or species may require additional optimization .
Note : Presence of TPB does not interfere with the enzymatic reaction .
Vortex mix thoroughly and pulse spin .
Amplify the samples in the thermal cycler using the following PCR cycling conditions : 95°C for 10 minutes ; 15 cycles of : 95°C for 5 seconds ; 60°C for 15 seconds ; 72°C for 1 minute ; 72°C for 5 minutes .
Hold at 4°C Safe Stopping Point : PCR products could be stored at - 20°C for up to seven days .
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 ) .
Note : See Appendix A for a more detail description of BioAnalyzer High Sensitivity DNA assay profile of the PCR products .
Note : The BioAnalyzer High Sensitivity DNA assay has a 10 % deviation on sizing accuracy .
The TailorMix Gel - Free miRNA Sample Preparation protocol enables the generation of micro RNA libraries form as low as 150ng Human kidney total RNA ( Figure 3 and Figure 4 ) .
However miRNA populations vary among different samples , the use of total RNA from other tissue or species may cause variations in PCR profiles .
Gel - free size selection is suitable for libraries which has a strong 140bp library product peak in compare to the 120bp artifact product peak .
It is recommended to use the PAGE - size selection approach for low yield libraries ( weak 140bp library product ) and libraries that have a strong 120bp artifact product peak .
See Table 2 in the Appendix for examples .
Gel - Free Library Purification Note : Sample volume may change after PCR .
To ensure purification efficiency , bring sample volume back to 30 μL before starting Gel - Free Purification steps if necessary .
Vortex the TailorMag Purification Beads ( TPB ) until they are evenly resuspended .
Prepare 80 % ethanol for rinse step .
Add TPB to each sample in the PCR tube according to the following table .
Votex mix thoroughly and pulse spin .
Incubate at room temperature for 5 minutes .
Note : Do NOT perform strong centrifugation because it will separate TPB from the sample .
Place the sample tube on the TailorMag PCR - tube magnetic stand at room temperature for 5 minutes , or until solution clears up .
DO NOT DISCARD SUPERNATANT .
Keep sample tube on the magnetic stand .
Transfer 55 μL of the clear supernatant to fresh sample tubes .
Note : Do not disrupt the TPB pellet .
Contamination of TPB pellet to the next step may affect final library quality .
Add TPB to clear supernatant from Step 27 .
Vortex mix thoroughly and pulse spin .
Incubate at room temperature for 5 minutes .
Note : Do NOT perform strong centrifugation because it will separate TPB from the sample .
Place the sample tube on the magnetic stand at room temperature for 5 minutes , or until solution clears up .
Remove and discard 60 μL of the supernatant .
Note : Sample recovery may be affected if the TPB pellet is disrupted .
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 .
Tip : Point pipette tip towards opposite direction as the TPB pellet .
Gently pipette the 80 % ethanol up and down once , then discard the rinse solution .
Air dry sample tube at room temperature .
Note : TailorMag Purification Beads are dried within 5 to 15 minutes at room temperature .
Proceed to Step 33 when the appearance of the TPB pellet turns form glossy / shiny ( wet ) to matte ( dry ) .
Sample recovery may be affected if beads are over - dried and appear powdery .
Remove sample tube from the magnetic stand .
Add 27 μL of TE buffer to the dried TPB pellet .
Vortex to resuspend and pulse spin .
Incubate resuspension at room temperature for 2 minutes .
Library Validation Use of an Agilent Technologies 2100 Bioanalyzer is recommended as a quality control analysis of your sample library .
Use 1 μL of resuspended library from Step 33 on a High Sensitivity DNA chip to check the size , purity and concentration of the sample .
Note : The BioAnalyzer High Sensitivity DNA assay has a 10 % deviation on sizing accuracy .
Note : If high percentage of 120bp peak remains , use PAGE size selection gel to extract the 140bp micro RNA library .
See Table 2 in the Appendix for references .
Figure 3 TailorMix Gel - Free miRNA libraries from Human Kidney Tissue Total RNABioAnalyzer High Sensitivity DNA assay profiles
