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
