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Home  |  Protocols  |  Molecular Biology  |  SELEX methodology for cell-specific aptamers for targeted therapies

Protocol - SELEX methodology for cell-specific aptamers for targeted therapies

Selex protocol for selection of GL21.T and Gint4.T and CL4 aptamers

Category:  Molecular Biology
Last revision: Apr 02, 2015
Author(s):  L.Cerchia, V. de Franciscis
Contact
Name: Laura Cerchia
Address: Institute of Experimental Endocrinology and Oncology “G. Salvatore” , CNR via Pansini 5, 80131 Naples Italy
Phone: +39 081 3722343
Email: l.cerchia@ieos.cnr.it

Steps

Description Temperature Time Note
Preparation of the 2’FPy-modified RNA library
PERFORM TRASCRIPTION
37°C
o.n.
FV=600 ul
●Recover trascription
●Treated with DNase I to remove contamination of ssDNA
●Add 10 units of DNase I and incubate
37°C
20 min
●Concentrate and desalt RNA transcriptions with phenol water/chloroform/isoamyl alcohol (25:24:1):
½ volume of Phenol water ½ volume of chloroform/isoamyl alcohol
●Vortex
●Centrifuge 13200 rpm
4°C
2 min
●Recover supernatant in a new 2mL tube
●Add 100ul of water to the original tube
●Vortex
●Centrifuge 13200 rpm
4°C
2 min
●Recover the supernatant and add it to the supernatant recovered before and add to this ½ volume of chloroform/isoamyl alcohol (24:1).
●Vortex
●Centrifuge 13200 rpm
4°C
2 min
●Add : 1/10 volume of NaAcetate 3M pH7 2 or 3 volume of ETOH 1.5 ul of linear acrylamide every 10ul of the recoverd supernatant volume
●Centrifuge 14000 rpm
4°C
30 min
●Suspend the pellet in 30 ul of loading solution and purify on 8% denaturing polyacrylamide gel using ; using as marker, a solution of xylene/cyanol.
RECOVER RNA
●Passively elute RNA from the gel in elution buffer (Elution buffer: 200mM NaAcetate, 2mM EDTA)
42°C
●Recover the eluted RNA following 2 h and precipitate at -20°C o.n. by adding 1/10 vol NaAc 3M and 2/3 vol ethanol;,
●Perform a second elution
42°C
o.n.
●Recover the second elution and precipitate. by adding 1/10 vol NaAc 3M and 2/3 vol ethanol and snap-cooled on dry ice and ethanol
●Centrifuge the first and the second elution at 14,000 rpm
4°C
30 min
●Suspend the pellet in H2O (20 - 40 ul) and quantified the RNA concentration
Store the sample at -20°C.
SELEX ROUND
●Incubate RNA (800-300 pmol) with cells
●Defrost RNA sample;
PERFORM DENATURATION/RENATURATION STEP
●Heat RNA (800 pmol) in 1.5. ml of DMEM serum free
5 min
85° C
●Snap-cooled on ice;
2 min
●Allowed to warm up to 37° C before incubation with the cells
10 min
PERFORM COUNTER SELECTION STEP (NON-TARGET CELLS, 2X 106-107
●Wash the cells 1x with 15ml DMEM serum free
●Add denaturated/renaturated RNA (1.5 ml) to 13.5 ml DMEM serum free (final volume 15ml)
●Incubate with non-target cells
30 min
37°C
● Recover the media containing the unbound RNAs (to be used for selection step)
●Lyse with 2 ml TRiZol reagent (to recover the bound of counter selection).
Store at -80°C
PERFORM SELECTION STEP (TARGET CELLS 2x106- 8x106
●Wash the cells 1x with 15ml DMEM serum free
●Add the recovered media containing unbound RNAs from counter selection step to the target cells (15 mL)
●Incubate
30 min
37°C
●Remove unbound RNAs (do not trash, you can retain it to check the unbound)
●Wash the cells 6x with 10ml DMEM serum free
●Lyse with 2 ml TRiZol reagent (bound of selection).
Store at -80°C overnight
At this point you can proceed with Trizol RNA extraction
●Defrost 2 ml TRiZol reagent (bound of selection) and Extract total RNA
●Reverse transcribe the RNA by using p20 rev primer.
PERFORM PCR MUTATION
●Program PCR mutation (10 cycles)
●Check the generation of the correct-sized PCR product by running a sample on a 2% agarose gel with a DNA size ladder. If a distinct band appears, proceed with PCR no mut. Otherwise, perform 5 additional cycles of PCR mut and repeat the gel.
The resulting PCR can be directly used for Transcription of RNA for the next round of SELEX.

 

Quality validation: Yes

Validation info

The protocol has been published in a peer-reviewed journal.

 

Citations
Esposito CL, Passaro D, Longobardo I, Condorelli G, Marotta P, Affuso A, de Franciscis V, Cerchia L.A neutralizing RNA aptamer against EGFR causes selective apoptotic cell death. PLoS One. 2011;6:e24071.
Cerchia L, Esposito CL, Camorani S, Rienzo A, Stasio L, Insabato L, Affuso A, de Franciscis V. Targeting Axl with an high-affinity inhibitory aptamer. Mol Ther. 2012;20:2291-303.
Cerchia L, Giangrande PH, McNamara JO, de Franciscis V. Cell-specific aptamers for targeted therapies. Methods Mol Biol. 2009;535:59-78.

 

Related Aptameres:

Solutions

  • Loading solution 500ul

    Reagents F-concentration Quantity I-concentration
    Deionized Formamide
    Formamide 95%
    475 ul
    Formamide 99,5%
    RNasi free water
    10 ul
    EDTA pH 8.0
    EDTA 0,01M pH 8.0
    10 ul
    EDTA 0,5M pH 8.0
    Bromophenol

    Note

    N.B Formamide oxidies readily in air and should be deionized by passage through a mixes-bed resin (Bio-Rad AG 501-X8) until its pH is neutral. It is then recrystallized at 0°C and stored at -20°C in small aliquots in tightly capped tubes

  • Transcription mix 600ul

    Reagents F-concentration Quantity I-concentration
    T7 Transcription buffer
    1X
    120 uL
    5X
    2'F-2'-dCTP
    1mM
    60 uL
    10mM
    2'F-2'-dUTP
    1mM
    60 uL
    10mM
    ATP
    1mM
    60 uL
    10mM
    GTP
    1mM
    60 uL
    10mM
    DTT
    16,7 mM
    100 uL
    100mM
    RNase inhibitors
    28 U/uL
    15 uL
    40 U/uL
    T7 RNA Pol.
    1,5 U/uL
    6 uL
    50 U/uL
    Inorganic Pyrophosphatase
    1 U/uL
    3 uL
    200U/uL
    Template
    0,5 pmol/uL
    Water
    to 600 uL

  • PCR mix 300ul

    Reagents F-concentration Quantity I-concentration
    Buffer PCR
    1X
    30 uL
    10X
    dNTP
    0,2 mM
    6 uL
    10mM
    Rev Primer
    0,2 uM
    0,6 uL
    100 uM
    Fw Primer
    0,2 uM
    0,6 uL
    100 uM
    TAQ
    0,05 U/uL
    5 uL
    5 U/uL
    Template
    60 ng
    Water
    to 300 uL

    Note

    N.B The quantity of MgCL2 is variable

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