Methods of releasing and analyzing cellular components
A system for analyzing a biological sample may include at least one sample acquisition stage comprising a sample acquisition device for acquiring the biological sample from a sample source; a droplet generator device for forming a droplet wrapped in an immiscible carrier fluid, wherein the wrapped droplet comprises at least the biological sample and a reagent, the droplet generator configured to receive the biological sample transferred from the sample acquisition device; a collection vessel for collecting the wrapped sample droplet from the droplet generator, the vessel configured to contain a carrier fluid for receiving and protecting the sample droplet; and an analysis system for analyzing the wrapped sample droplet and detecting products of a polymerase chain reaction.
1. A method comprising:
controlling fluid flow through one or more channels to generate aqueous droplets wrapped in a carrier fluid that is immiscible with the aqueous droplets, wherein the aqueous droplets comprise a subset of droplets, each droplet of the subset comprising at least one cell, and a lysis reagent;
lysing the at least one cell in each droplet of the subset of droplets to release internal cellular components comprising RNA;
dispensing the RNA of the subset of droplets released from the lysing to a vessel comprising one or more reservoirs; and
while in the vessel, collectively subjecting the RNA of the subset of droplets released from the lysing to a reverse transcription assay to produce cDNA from the RNA.
2. The method according to claim 1 , wherein the at least one cell in each aqueous droplet of the subset of droplets is derived from solid tissue.
3. The method of claim 1 , wherein the at least one cell in each aqueous droplet of the subset of droplets is an adherent cell.
4. The method according to claim 1 , wherein the at least one cell in each aqueous droplet of the subset of droplets is a single cell.
5. The method according to claim 1 , wherein dispensing the RNA to the vessel comprises dispensing the subset of droplets containing the RNA released from the lysing to the vessel.
6. The method according to claim 5 , wherein the dispensing the subset of droplets occurs via siphoning the subset of droplets.
7. The method according to claim 1 , wherein:
controlling the fluid flow through the one or more channels to generate the aqueous droplets comprises flowing a sample fluid comprising a plurality of cells and the lysing reagent through different channels to a fluidic junction comprising the carrier fluid, and
at the fluidic junction, the aqueous droplets wrapped in the carrier fluid are generated.
8. The method according to claim 7 , further comprising flowing a reverse transcriptase through a channel of the different channels to the fluidic junction for incorporation into the aqueous droplets.
9. The method according to claim 1 , wherein the at least one cell in each aqueous droplet of the subset of droplets is from a human tissue or a body fluid.
10. The method according to claim 1 , wherein the at least one cell in each aqueous droplet of the subset of droplets is a tumor cell.
11. The method of claim 1 , wherein:
controlling the fluid flow through the one or more channels to generate the aqueous droplets comprises flowing a sample fluid comprising a plurality of cells and the lysing reagent through a common channel to a fluidic junction comprising the carrier fluid, and
at the fluidic junction, the aqueous droplets wrapped in the carrier fluid are generated and the cells are mixed with the lysing reagent resulting in the lysis of the cells.
12. The method of claim 1 , further comprising imaging the droplets of the subset of droplets in a channel of the one or more channels.
13. The method of claim 1 , further comprising rupturing the droplets of the subset of droplets prior to dispensing the RNA into the vessel.
14. The method of claim 1 , further comprising rupturing the droplets of the subset of droplets in the vessel.
15. The method of claim 1 , wherein each droplet of the subset comprises a reverse transcriptase.
16. The method of claim 1 , further comprising amplifying the cDNA by a polymerase chain reaction in the vessel.
17. The method of claim 16 , further comprising detecting a polymerase chain reaction product in the vessel.
18. The method of claim 1 , wherein the vessel comprising the one or more reservoirs is chosen from a multi-well plate and a tube.
19. The method of claim 1 , wherein the vessel comprises a multi-well plate, the one or more reservoirs comprise one or more wells, and the dispensing comprises dispensing one or more droplets of the subset into the one or more wells.
20. The method of claim 1 , wherein the at least one cell in each droplet of the subset of droplets comprises a mammalian cell.