OPTIMIZE YOUR SAMPLE PREP PROTOCOL Precellys Webinar

Transcription

1 OPTIMIZE YOUR SAMPLE PREP PROTOCOL Precellys Webinar

2 YOUR SPEAKERS TODAY ARE 2 Stéphane Karoubi Product Manager Sophie Dubacq Application Specialist

3 AGENDA 3 1. Sample Preparation 1. Traditional methods 2. Bead-Beating method 2. Sample prep optimization 1. Lysing kits choice 2. Ask the right questions 3. Choose the right kit 3. Applications & case studies 4. Conclusion

4 SAMPLE PREPARATION

5 TRADITIONAL METHODS 5 Sonicator Mortar & pestel Blaid mixer Unavoidable cleaning step Poor reproducibility Low throughput Low flexibility

6 BEAD-BEATING METHOD 6 Bead-beating s answers to sample preparation needs: Reproducibility & yield Adaptable to any biological sample Time saving, multiple samples at once Cross-contamination free, single use consumables Extraction quality & quantity improvement

7 7 SAMPLE PREP OPTIMIZATION Ask the right questions

8 CHOOSE THE RIGHT LYSING KITS 8 4 Volumes: 0.5, 2, 7 & 15mL Sample type? Soft: Animal tissue or plant Microorganisms Elastic Hard/dry material CERAMIC GLASS COMPLEX MATRIX METAL Liver, brain, leaves, organs, etc. Bacteria, yeast, spores, cells, viruses Skin, cartilage, innervated tissue, etc. Seeds, teeth, hair, roots, tumors, etc.

9 ASK QUESTIONS 9 Sample size/volume/weight? < 20mg or 200µL From 20mg to 200mg < 1.6mL From 200mg to 2g < 6mL From 2g to 4g < 9mL 0.5mL 2mL 7mL 15mL

10 ASK QUESTIONS 10 Thermo-sensitive molecule? RNA? Proteins? Need a cooling solution Before, during and after homogenization Step on ice Cooling unit

11 CHOOSE THE RIGHT LYSING KIT

12 PLANT OR ANIMAL TISSUE 12 Sample type: SOFT Kidney, liver, lung, brain Leaves Sample type: HARD Tumor, muscle, Some leaves Sample type: ELASTIC Skin, artery, cartilage Ceramic beads of 1.4mm Ceramic beads of 2.8mm or more Metal beads of 2.8mm Mix of Ceramic beads (1.4 & 2.8mm or 2.8 & 5.0mm)

13 VERY HARD SAMPLES 13 Beads available Metal beads of 2.8mm HAIR Metal beads of 2.8mm RICE/SEEDS Ceramic beads of 6.8mm Ceramic beads of 6.8mm Mix of Ceramic beads ( mm) Result : Result :

14 MICROORGANISMS 14 BACTERIA (Gram + or -) E. Coli, Mycobacterium, Staphylococcus, TOUGH Fungi, Spores, Viruses INSIDE SAMPLE Feces, leaves, tissues, water, soil, Glass beads of 0.5 or 0.1mm Glass beads of 0.5mm Mix of ceramic (1.4) and glass (0.1) beads

15 PRECELLYS FAMILY OF PRODUCTS A COMPLETE HOMOGENIZATION SOLUTION 15 Multiple applications High throughput and flexibility High throughput Routine work Flexible Affordable Cooling option Protects sensitive molecules 27 different lysing kits, Empty tubes & beads in bulk

16 APPLICATION & CASE STUDY

17 Response (Cts/ g) WHOLE IRIS-CILIARY BODY PORK LYSIS 17 CONTEXT Whole iris-ciliary body pork ceramic beads The Minilys has a max speed of 5000 rpm, use stronger lysing matrix CKMix50 PROTOCOL Homogenization of whole iris-ciliary body into 7mL vial with Minilys 0,7g 7mL CKMix50 7mL : 2.8 and 5.0mm ceramic beads metabolite extraction 5000 rpm, 4x30 sec (15 sec break) 1,00E+07 1,00E+06 1,00E+05 1,00E+04 1,00E+03 Precellys Average Minilys Avera RESULTS Minilys can meet Precellys efficiency on difficult samples using the right lysing kit 1,00E+02 1,00E+01 1,00E+00 Precellys - E1E2 Minilys - E1e2 Figure 1. LC-MS/MS analysis of the target analyte after Precellys and Minilys homogenization

18 SKIN GRINDING 18 CONTEXT Deramatomed human abdominal skin ceramic or metal beads PROTOCOL < 300 mg - 2mL MK28 2mL: 2,8mm metal beads Compound extraction Precellys rpm, 1x30 (dermis), 2x60 (epidermis) sec (30 sec break) RESULTS Precellys procedure showed increased extraction of compound SB from the dermis & epidermis samples Higher efficiency Dermal (A) and epidermal (B) amount (µg) of SB after a 15 and 48 hour liquid extraction (LE) versus homogenization using the Precellys

19 BACTERIAL LYSIS 19 CONTEXT E. coli cells normalized to an OD600= 50 / 1mL of culture glass beads SDS-PAGE + Coomassie blue staining PROTOCOL 1 ml - 2mL VK01 2mL : 0.1mm glass beads Protein extraction Precellys Evolution 9000 rpm, 6x30 sec in the cold room (60 sec break) RESULTS Microbiology application with Precellys high protein extraction quantity/quality with the presence of strong bands Figure 1. Total soluble protein profiles after Precellys homogenization using VK01 glass beads (Lanes 2 and 3)

20 CELL DISSOCIATION 20 CONTEXT Mouse whole lung ceramic beads Cell count and viability (Trypan blue) PROTOCOL 0,3 gram - choose 7mL CK28 7mL : 2.8 mm ceramic beads Isolation of immune cells from mice lungs Precellys Evolution Flow cytometry Sieve Precellys 4500 rpm, 1x10 sec RESULTS The Precellys Evolution leads to a higher yield of some immune cells (CD45+), notably neutrophils and lymphocytes.

21 CONCLUSION

22 HOW PRECELLYS TEAM CAN HELP YOU? 22 Application notes Protocols Application Center Application specialist Validated protocols by users Optimized protocols Material & method information Results Targeted molecule Lysing kit choice Buffer advice Parameters Used downstream analysis Find validated protocols Compare protocols to others Work by key words Direct contact Support for optimization Comfort protocol choice Any application questions

23 CONCLUSION 23 Sample prep optimization Use the kits theory Use online resources Adjust parameters Contact Precellys team Define the type of sample Find the right volume of tube Find the right bead mix Find the right bead mix Comfort your choice Find validated protocols Results Optimize results Play on speed, cycles, time Change lysing kits Optimize results Direct contact Any application questions

24 THANK YOU FOR YOUR ATTENTION Feel free to ask any question with chat box